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2. PROTEINS
Contents
1. Proteins: Structure, Function & Evolution
i. 3D protein structure & Motifs
ii. Protein Sorting & Transport
iii. Protein folding & degradation
2. Protein-Nucleic acid Interactions

Protein Sorting & Transport (localization/
Trafficking)
The best-characterized targeting system begins in the
ER
Most lysosomal, membrane, or secreted proteins have
an amino-terminal signal sequence that marks them
for translocation into the lumen of the ER
Hundreds of such signal sequences have been
determined
The carboxyl terminus of the signal sequence is defined
by a cleavage site - protease action removes the
sequence after the protein is imported into the ER

Proteins are synthesized in two types of location:
1. Ribosomes in the cytosol - synthesize the vast
majority of proteins
2. Ribosomes within organelles (mitochondria or
chloroplasts) - synthesize a small minority of proteins
Proteins synthesized in the cytosol can be divided into
two general classes with regard to localization:
1. Proteins associated with membranes
2. Proteins not associated with membranes

Each class can be subdivided further, depending on
- whether the protein associates with a particular
structure in the cytosol or with a particular membrane

The cytoplasm contains a series of membranous bodies,
including endoplasmic reticulum (ER), Golgi apparatus,
endosomes, and lysosomes
This is sometimes referred to as the "reticuloendothelial system."
Proteins that reside within these compartments are inserted into
ER membranes, and then are directed to their particular
locations by the transport system of the Golgi apparatus.
Proteins that are secreted from the cell are transported to the
plasma membrane and then must pass through it to the exterior.
They start their synthesis in the same way as proteins
associated with the reticuloendothelial system, but pass entirely
through the system instead of halting at some particular point
within it.

The cell has the following possible ultimate
destinations for a newly synthesized protein:
1. Cytosolic (or "soluble") proteins - are not localized
in any particular organelle
- They are synthesized in the cytosol, and remain there,
where they function as individual catalytic centers, acting
on metabolites that are in solution in the cytosol
2. Macromolecular structures - may be located at
particular sites in the cytoplasm
Ex. centrioles are associated with the regions that
become the poles of the mitotic spindle

3. Nuclear proteins - must be transported from their site
of synthesis in the cytosol through the nuclear envelope
into the nucleus
4. Membrane proteins - Most of the proteins in
cytoplasmic organelles are synthesized in the cytosol
and transported specifically to (and through) the
organelle membrane
- Those proteins that are synthesized within the
organelle remain within it

Overview: proteins that are localized post-translationally are
released into the cytosol after synthesis on free ribosomes. Some
have signals for targeting to organelles such as the nucleus or
mitochondria. Proteins that are localized cotranslationally
associate with the ER membrane during synthesis, so their
ribosomes are “membrane-bound.” The proteins pass into the
endoplasmic reticulum, along to the Golgi, and then through the
plasma membrane, unless they have signals that cause retention
at one of the steps on the pathway. They may also be directed to
other organelles, such as endosomes or lysosomes.

Overview:
• Post-translational---Cytosol---free ribosome---targeted to
organelles (Mitochondria, nucleus, chloroplasts)
• Co-translational-----ER----------Membrane-bound ribosomes-----
targeted for secretory pathway (ER----Golgi----Membrane
coated proteins (COPs)----Plasma membrane, Endosomes or
lysomes) unless have retention signals for ER and Golgi

Section 12-4. Membr
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(a)

Figure 12-58. Post-translational processing of proteins. Proteins
destined for secretion, insertion into the plasma membrane, or
transport to lysosomes are synthesized by RER-associated
ribosomes (blue dots; top). As they are synthesized, the
proteins (red dots) are either translocated into the lumen of the
ER or inserted into its membrane. After initial processing in the
ER, the proteins are encapsulated in vesicles that bud off from
the ER membrane and subsequently fuse with the cis Golgi
network. The proteins are progressively processed in the cis,
medial, and trans cisternae of the Golgi. Finally, in the trans
Golgi network (bottom), the completed glycoproteins are sorted
for delivery to their final destinations, the plasma membrane,
secretory vesicles, or lysosomes, to which they are transported
by yet other vesicles.

The process of inserting into or passing through a
membrane
The protein presents a hydrophilic surface, but the
membrane is hydrophobic
Like oil and water, the two would prefer not to mix
Solution: a special structure in the membrane
through which the protein can pass---Translocon (Sec
proteins; Sec 61 and Sec Y)
Protein Translocation

There are three different types of arrangement for
translocon structures
1. Direct interaction between the translocon and
the substrate protein---ER, Mitochondria,
chloroplasts
2. Carrier---Peroxisosmes
3. Carrier via pores---Nuclear pore

1. Proteinaceous structures embedded in their
membranes - ER, mitochondria, and chloroplasts
- allow proteins to pass through without contacting the
surrounding hydrophobic lipids
P r o t e i n p a s s e s
through translocon
A substrate protein
binds directly to the
s t r u c t u r e , i s
transported by it to the
other side, and then
released.

2. Peroxisomes also have such structures in their
membranes, but the substrate proteins do not bind
directly to them.
the protein
al contact with a
cation apparatus
This called co-
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he cytosol when
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ith macromolec-
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quence motif that
r to be assembled
ins released from
rom the presence
Proteins are transported into peroxisomes by a carrier protein
that binds them in the cytosol, passes with them through the
membrane channel, and releases on the other side

3. The nuclear pore
Employed for transport into the nucleus
Included in this apparatus are carrier proteins that
bind to the substrates and transport them through
the pore to the other side

Proteins enter the nucleus by
passage through very large
nuclear pores. The transport
apparatus is distinct from the
pore itself and includes
components that carry the
protein through the pore

Protein translocation may be:
• Post-translational or
• Co-translational
There are two ways for a protein to make its initial
contact with a membrane:
1. Cotranslational translocation - The nascent protein
associate with the translocation apparatus while it is still
being synthesized on the ribosome
- used for proteins that enter the endoplasmic
reticulum

2. Post-translational translocation - The protein
released from a ribosome after translation has been
completed
- Then the completed protein diffuses to the
appropriate membrane and associates with the
translocation apparatus

The free ribosomes synthesize all proteins except those
that are translocated co-translationally
The proteins are released into the cytosol when their
synthesis is completed and then:
1. Some of these proteins remain free in the cytosol in
soluble form
2. Others associate with macromolecular cytosolic
structures - filaments, microtubules, centrioles, etc.,
or
2.1. Transported to the nucleus, or
2.2. Associate with membrane bound organelles by post-
translational translocation—Mitochondria, chloroplast

Proteins that reside within the reticuloendothelial system
enter the endoplasmic reticulum while they are being
synthesized
The principle of co-translational translocation:
- the nascent protein is responsible for recognizing the
translocation apparatus
This requires the signal for co-translational translocation
to be part of the protein that is first synthesized: it is
usually located at the N-terminus

The N-terminal sequence comprises a leader that is not
part of the mature protein
The protein carrying this leader is called - a preprotein
It is a transient precursor to the mature protein
The leader is cleaved from the protein during protein
translocation

Proteins can enter the ER Golgi pathway only by associating with
the ER while they are being synthesized.

Signal sequences initiate translocation
• Co-translational insertion is directed by a signal
sequence
• Usually this is a cleavable leader sequence of 15-30
N-terminal amino acids
• At or close to the N-terminus are several polar
residues, and within the leader is a hydrophobic core
consisting exclusively or very largely of hydrophobic
amino acids

• A ribosome starts synthesis of a protein without
knowing whether the protein will be synthesized in the
cytosol or transferred to a membrane
• It is the synthesis of a signal sequence that causes
the ribosome to associate with a membrane
The signal sequence of bovine growth hormone consists of the N-terminal 29
amino acids and has a central highly hydrophobic region, preceded or flanked
by regions containing polar amino acids.

Feature of signal sequences
Signal sequences: 13 to 36 amino acid residues
All have the following features:
1. About 10 to 15 hydrophobic amino acid;
2. ≥ 1 +vely charged residues - usually near the N
terminus, preceding the hydrophobic sequence; and
3. A short sequence at the C-terminus (near the
cleavage site) - relatively polar, typically having
amino acid residues with short side chains (especially
Ala) at the positions closest to the cleavage site

Translocation into the ER directed by amino-terminal signal
sequences of some eukaryotic proteins. The hydrophobic core (yellow)
is preceded by one or more basic residues (blue). Note the polar and short-
side-chain residues immediately preceding (to the left of, as shown here)
the cleavage sites (indicated by red arrows).

2-47 N-Terminal sequences of some eukaryotic
preproteins. The hydrophobic cores (brown) of most
Bovine growth hormone
Bovine proalbumin
Human proinsulin
Human interferon γ
Zea mays rein protein 22.1
Human α-fibrinogen
Human IgG heavy chain
Rat amylase
Murine α-fetoprotein
Chicken lysozyme
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Signal
peptidase
cleavage
site
signal peptides are preceded by basic residues (blue).[After
Watson,M.E.E.,NucleicAcids Res. 12, 5147–5156 (1984).]
N-Terminal sequences of some eukaryotic secretory
preproteins. The hydrophobic cores (brown) of most signal
peptides preceded by basic residues (blue).

• To associate with a membrane (or any other type of
structure), a protein requires an appropriate signal
• A sequence motif that causes it to be recognized by
a translocation system (or to be assembled into a
macromolecular structure)
Signals used by proteins released from cytosolic
ribosomes:
• "nuclear localization signals" – determine import
into the nucleus
• A variety of rather short sequences within proteins
(internal signal, sequences within the protein)
• These enable the proteins to pass through nuclear
pores

• One type of signal that determines transport to
the peroxisome is a very short C-terminal
sequence
• Mitochondrial and chloroplast proteins – require
N-terminal sequences of ~25 amino acids in
length that are recognized by receptors on the
organelle envelope

Proteins synthesized on free ribosomes in the cytosol are
directed after their release to specific destinations by short
signal motifs

How substrate proteins associate with membranes?
Protein translocation can be divided into two general
stages:
1. Ribosomes carrying nascent polypeptides associate
with the membranes;
2. The nascent chain is transferred to the channel and
translocates through it
• The attachment of ribosomes to membranes
requires the signal recognition particle (SRP)
• The signal sequence interacts with the SRP
(signal recognition particle)

• Ribosomes
• ER
• Signal sequences
• SRP (signal recognition particle)
• SRP receptor
• Ribosome receptor
• Peptide translocation complex: Signal peptidase
• GTPase (Molecular Switch): GTP----GDP + P
Protein translocation

cytosol
E
R
1.The targeting pathway begins with initiation of protein
synthesis on free ribosomes
Translocation pathway of membrane proteins:

cytosol
E
R
2. The signal sequence appears early in the synthetic process,
because it is at the amino terminus, which is synthesized first

cytosol
ER
3. As it emerges from the ribosome, the signal sequence—and the ribosome
itself—are bound by the large signal recognition particle (SRP);
- SRP then binds GTP and halts elongation of the polypeptide when it is about
70 amino acids long and the signal sequence has completely emerged from
the ribosome

cytosol
ER
4. The GTP-bound SRP now directs the ribosome (still bound to the mRNA)
and the incomplete polypeptide to GTP-bound SRP receptors in the cytosolic
face of the ER
- the nascent polypeptide is delivered to a peptide translocation complex
in the ER, which may interact directly with the ribosome

cytosol
ER
5. SRP dissociates from the ribosome, accompanied by
hydrolysis of GTP in both SRP and the SRP receptor

cytosol
ER
6. Elongation of the polypeptide now resumes, with the GTP-driven
translocation complex feeding the growing polypeptide into the ER lumen
until the complete protein has been synthesized

cytosol
ER
7. The signal sequence is removed by a signal peptidase within the ER
lumen
8. The ribosome dissociates and is recycled

Directing eukaryotic proteins with the appropriate signals
to the endoplasmic reticulum. This process involves the
SRP cycle and translocation and cleavage of the nascent
polypeptide. The steps are described in the text. SRP is a rod-
shaped complex containing a 300 nucleotide RNA (7SL-RNA)
and six different proteins (combined Mr 325,000). One protein
subunit of SRP binds directly to the signal sequence, inhibiting
elongation by sterically blocking the entry of aminoacyl-tRNAs
and inhibiting peptidyl transferase. Another protein subunit
binds and hydrolyzes GTP. The SRP receptor is a heterodimer
of (Mr 69,000) and (Mr 30,000) subunits, both of which bind
and hydrolyze multiple GTP molecules during this process.

• The SRP and SRP receptor function catalytically to transfer a
ribosome carrying a nascent protein to the membrane
• The first step is the recognition of the signal sequence by the
SRP
• Then the SRP binds to the SRP receptor and the ribosome
binds to the membrane on ribosome receptor
The SRP has two important abilities:
1. It can bind to the signal sequence of a nascent
secretory protein
2. It can bind to a protein (the SRP receptor) located
in the membrane of ER

• The signal peptide is cleaved from a translocating
protein by a complex of 5 proteins called the
signal peptidase
• It is located on the lumenal face of the ER
membrane, which implies that the entire signal
sequence must cross the membrane before the
cleavage event occurs
• Homologous signal peptidases can be
recognized in eubacteria, archaea, and
eukaryotes.

Ribosomes synthesizing secretory proteins are attached to the membrane via
the signal sequence on the nascent polypeptide
Stages of translocation of membrane proteins

What is SRP?
The SRP is a ribonucleoprotein complex (11S):
- 6 proteins (total mass 240 kD) and
- a small 7S RNA (305 base, 100 kD)
The 7S RNA provides the structural backbone of the
particle
- the individual proteins do not assemble in its absence
The 7S RNA of the SRP particle is divided into two parts:
- the Alu domain: 154 bases
- the S domain: 151 bases

The two domains of the 7S RNA of the SRP are defined by its relationship to
the Alu sequence. Five of the six proteins bind directly to the 7S RNA. Each
function of the SRP is associated with a particular protein (s).

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Figure 12-48 Sequence and secondary structure of canine 7S
RNA. Its various double helical segments (denoted H1 through
H8) and loops (denoted L1 and L1.2), are drawn in red and
yellow with Watson–Crick base pairs represented by connecting
lines and non-Watson–Crick base pairs indicated by dots. The
positions at which the various SRP proteins bind to the 7S RNA
are indicated in cyan, blue, and gray. [Courtesy of Roland
Beckmann, Humboldt University of Berlin, Germany.]
Sequence and secondary structure of canine 7S RNA. Its
various double helical segments (denoted H1 through H8) and
loops (denoted L1 and L1.2), are drawn in red and yellow with
Watson–Crick base pairs represented by connecting lines and
non-Watson–Crick base pairs indicated by dots. The positions at
which the various SRP proteins bind to the 7S RNA are
indicated in cyan, blue, and gray.

Different parts of the SRP structure have separate
functions in protein targeting
SRP54 can be cross linked to the signal sequence of a
nascent protein; it is directly responsible for recognition
of the substrate protein—Signal recognition
The SRP68-SRP72 dimer binds to the central region of
the RNA; it is needed for recognizing the SRP receptor
—SRP receptor binding
The SRP9-SRP14 dimer binds at the other end of the
molecule; it is responsible for elongation arrest

SRP receptor
The SRP receptor in mammals is a dimer containing
subunits SRα (72 kD) and SRβ (30 kD) which are
GTPases
The β subunit is an integral membrane protein to
which α subunit bound
The amino-terminal end of the large α subunit is
anchored by the β subunit.
The bulk of the a protein protrudes into the cytosol

SRP receptor
A large part of the sequence of the cytoplasmic region
of the protein resembles a nucleic acid-binding protein,
with many positive residues
This suggests the possibility that the SRP receptor
recognizes the 7S RNA in the SRP

• GTP hydrolysis plays an important role in inserting the
signal sequence into the membrane
• Both the SRP and the SRP receptor have GTPase
capability
• The signal-binding subunit of the SRP, SRP54, is a
GTPase
• Both subunits of the SRP receptor are GTPases
• All of the GTPase activities are necessary for a
nascent protein to be transferred to the membrane
• The ribosome then stimulates replacement of the
GDP with GTP

The SRP carries GDP when it binds the signal sequence. The
ribosome causes the GDP to be replaced with GTP.
• The signal sequence inhibits hydrolysis of the GTP
• This ensures that the complex has GTP bound
when it encounters the SRP receptor

• For the nascent protein to be transferred from the
SRP to the membrane, the SRP must be released
from the SRP receptor
• This requires hydrolysis of the GTPs of both the SRP
and the SRP receptor
The SRP and SRP receptor both hydrolyze GTP when the signal
sequence is transferred to the membrane.

• GTP binding to SRP54 (Ffh) and SRα (FtsY) is a prerequisite
for complex formation between SRP and SR, and GTP
hydrolysis leads to the dissociation of the SRP/SR complex

J. Luirink, I. Sinning / Biochimica et Biophysica Acta 1694 (2004) 17–35

ntial for SR function [28]. SRa is the ever, if this would be mainly a matter of anchoring, an
esentation of SRP and SR components in the three kingdoms of life: from Eubacteria on the top to Eukaryotes (e.g. human SRP) on the
ode is blue (Ffh/SRP54; NG domain in dark blue, M domain in light blue), cyan (SRP19), grey (SRP68/72), green (SRP9/14), black
ifs of the RNA (e.g. helices 2 –8; domain IV) which are referred to in the text are marked. The SRP receptor is shown in pink (FtsY, SRa;
nk, A domain in dark pink) and violet (SRh).
• T h e S R P c o r e
(SRP54/helix 8 of
the SRP RNA) is the
only part of SRP that
is present in all
SRPs
SRP receptor
Eukaryotes
• SRPα
• SRPβ
Prokaryotes
• FtsY
• H o m o l o g o u s t o
SRPα

SRP54
• The SRP core (SRP54/helix 8 of the SRP RNA) is the only
part of SRP that is present in all SRPs
• SRP54 (Ffh) is a multidomain protein and consists of a C-
terminal M domain and a so-called NG domain at the N-
terminus
• The M domain contains the binding sites for the SRP RNA
and the signal peptide, and
• The N and G domains comprise the conserved GTPase for
regulation of protein transport and for the interaction with the
SRP receptor

Where assembly of SRP takes place
• In eukaryotes, the assembly of SRP takes place in two
cellular compartments:
• the SRP RNA is transcribed in the nucleus whereas the
protein components are synthesized in the cytoplasm and
are then imported into the nucleus (except SRP54)
• They assemble on the SRP RNA into a pre-SRP, which is
then exported to the cytoplasm
• Binding of SRP54 completes the assembly of SRP
• Interestingly, the presence of SRP19 is a prerequisite for
binding of SRP54

• The translocon forms a pore
The translocon - the channel through the membrane
• Proteins that are part of the ER membrane form an
aqueous channel through the bilayer
• A translocating protein moves through this channel,
interacting with the resident proteins rather than with
the lipid bilayer

• The channel is sealed on the lumenal side - to stop
free transfer of ions between the ER and the cytosol
The translocon is a trimer of Sec61 (Sec61α, β, γ) that forms
a channel through the membrane. It is sealed on the lumenal
(ER) side

Sec61 is a trimeric membrane protein complex that is
structurally and functionally highly conserved
throughout all domains of life, known as
SecYEG in bacteria and
SecYEβ in archaea

Components of the translocon
1. Sec61 is the major component of the translocon
• consists of three transmembrane proteins: Sec61α, β,
γ
2. TRAM (Translocating associating membrane
protein)

Components of the translocon
• Have been identified in two ways:
• Resident ER membrane proteins that are cross linked
to translocating proteins are potential subunits of the
channel
1. sec mutants in yeast - named because they fail to
secrete proteins
- cause precursors of secreted or membrane
proteins to accumulate in the cytosol
These approaches identify the Sec61 complex:
- consists of three transmembrane proteins: Sec61α, β, γ
Sec61 is the major component of the translocon

2. A detergent provides a hydrophobic milieu that
mimics the effect of a surrounding membrane
In detergent, Sec61 forms cylindrical oligomers with a
diameter of ~85 A and a central pore of ~20 A
Each oligomer consists of 3-4 heterotrimers

Is the channel a preexisting structure or
might it be assembled in response to the association of a
hydrophobic signal sequence with the lipid bilayer?
Channels can be detected by their ability to allow the passage
of ions - measured as a localized change in electrical
conductance – (Patch clamp)
Ion-conducting channels can be detected in the ER
membrane, and
- their state depends on protein translocation
→ the channel is a permanent feature of the membrane

How translocon works; (opened and closed)?
• A channel opens when a nascent polypeptide is
transferred from a ribosome to the ER membrane
• The translocating protein fills the channel completely,
so ions cannot pass through during translocation
• If the protein is released by treatment with
puromycin, then the channel becomes freely
permeable
• If the ribosomes are removed from the membrane,
the channel closes
- suggests that the open state requires the presence of
the ribosome
- suggests that the channel is controlled in response to the
presence of a translocating protein

Measurements of the abilities of fluorescence
quenching agents of different sizes to enter the
channel
- suggest that it is large - with an internal diameter of
40-60 A

A nascent protein is transferred directly from the ribosome to the translocon.
The ribosomal seals the channel on the cytosolic side.

The translocon can be used by translocating proteins
in several ways:
1. It is the means by which nascent proteins are
transferred from cytosolic ribosomes to the lumen of
endoplasmic reticulum
2. It is also the route by which integral membrane
proteins of the ER system are transferred to the
membrane
3. Reverse translocation - proteins can be transferred
from the ER back to the cytosol

Translocation requires insertion into the translocon
and (sometimes) a ratchet in the ER
• The translocon and the SRP receptor are the basic
components required for co-translational translocation
• When the Sec61 complex is incorporated into artificial
membranes together with the SRP receptor, it can
support translocation of some nascent proteins
• Other nascent proteins require the presence of an
additional component, TRAM (Translocating
associating membrane protein)
- It is a major protein that becomes cross linked to a
translocating nascent chain
TRAM stimulates the translocation of all proteins

Translocation requires the translocon, SRP, SRP receptor, Sec61, TRAM,
and signal peptidase.

Post-translational insertion of a protein into a
membrane
• A more complex apparatus is required
• The same Sec61 complex forms the channel
Additional requirements:
1. Four other Sec proteins are also required
Sec62-63, Sec71 and Sec72
1. The chaperone BiP (Binding protein) and
2. Supply of ATP are required on the lumenal side of
the membrane

• In the absence of BiP, Brownian motion allows the
protein to slip back into the cytosol
• BiP grabs the protein as it exits the pore into the
endoplasmic reticulum
• This stops the protein from moving backward
The reason why BiP is required for post-translational
translocation but not for co-translational translocation
may be that:
- a newly synthesized protein is continuously
extruded from the ribosome and therefore cannot
slip backward

Bip acts as a ratchet to prevent backward diffusion of a translocating protein

• In the post-translational mode, Sec61 forms a stable
complex with the dimeric Sec62–Sec63 complex,
and in fungi, additionally with Sec71 and Sec72
• These accessory proteins facilitate the transient
binding of chaperones, in particular heat-shock 70
(Hsp70) family proteins, to the cytosolic and luminal
side of the ER post-translocon
• Hsp70 family proteins prevent misfolding of
translocated substrates in the ER lumen

Reverse translocation
• Sends proteins back to the cytosol for
degradation
The ER provides a "quality control" system
• Misfolded proteins are identified and degraded
• However, the degradation does not occur in the ER
• Require the protein to be exported back to the
cytosol

• Ubiquitination and proteasomal degradation both
occur in the cytosol (with a minor proportion in the
nucleus)
• The first indication that ER proteins are degraded in
the cytosol and not in the ER itself was provided by
evidence for the involvement of the proteasome
• The proteasome - degrades ubiquitinated proteins
• Inhibitors of the proteasome prevent the
degradation of aberrant ER proteins

Reverse translocation
• Transport from the ER back into the cytosol by a
reversal of the usual process of import
• The Sec61 translocon is used
• The translocon is not associated with a ribosome
Some mutations in Sec61 - prevent reverse
translocation, but do not prevent forward translocation
This could be either because
• There is some difference in the process or
• More likely because these regions interact with
other components that are necessary for reverse

• How the channel is opened to allow insertion of the
protein on the ER side – is not known
Special components are presumably involved
A model:
• Misfolded or misassembled proteins are
recognized by chaperones - transfer them to the
translocon

Proteins reside in membranes by means of
hydrophobic regions
An integral membrane protein - requires disruption of
the lipid bilayer in order to be released from the
membrane
Such proteins have at least one transmembrane
domain
- consist of an α-helical stretch of 21-26 hydrophobic
amino acids

Hydropathy (hydrophobicity) plot
Used to identify a sequence that fits the criteria for
membrane insertion
- measures the cumulative hydrophobicity of a
stretch of amino acids
A transmembrane protein - has domains exposed on
both sides of the membrane

The association of a protein with a membrane takes
several forms
The topography of a membrane protein depends on the
number and arrangement of transmembrane regions
A protein with a single transmembrane region
- its position determines how much of the protein is
exposed on either side of the membrane
A protein may have extensive domains exposed on both
sides of the membrane or
- may have a site of insertion close to one end, so that
little or no material is exposed on one side

Proteins with a single transmembrane domain fall into
two classes:
Group I proteins - the N-terminus faces the
extracellular space
- are more common
Group II proteins - the N-terminus faces the
cytoplasm

G r o u p I a n d g r o u p I I
transmembrane proteins have
opposite orientations with
regard to the membrane.
Two types of protein span the
membrane once

Orientations for proteins that have multiple
membrane-spanning domains
An odd number - both termini of the protein are on
opposite sides of the membrane
An even number - the termini are on the same face
Domains at either terminus may be exposed, and
internal sequences between the domains "loop out"
into the extracellular space or cytoplasm
Ex.
the 7-membrane passage or "serpentine" receptor;
the 12-membrane passage component of an ion
channel

The orientations of the
t e r m i n i o f m u l t i p l e
membrane spanning
proteins depends on
whether there is an odd or
e v e n n u m b e r o f
transmembrane segments
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to delete the image and then insert it again.

Does a transmembrane domain itself play any role in
protein function besides allowing the protein to insert into
the lipid bilayer?
simple group I or II proteins - it has little or no
additional function
- it can be replaced by any other transmembrane
domain
Transmembrane domains play an important role in the
function of proteins that make multiple passes through
the membrane or that have subunits that oligomerize
within the membrane

Polar regions in the membrane-spanning domains do
not interact with the lipid bilayer, but instead interact
with one another
- form a polar pore or channel within the lipid bilayer
Interaction between such transmembrane domains can
create a hydrophilic passage through the
hydrophobic interior of the membrane
- allow highly charged ions or molecules to pass through the
membrane - function of ion channels and transport of ligands
The transmembrane domains in such cases often
contain polar residues, which are not found in the
single membrane-spanning domains of group I and
group II proteins

Certain receptors that bind lipophilic ligands
- conformation of the transmembrane domains is
important
- the transmembrane domains (rather than the
extracellular domains) bind the ligand within the plane
of the membrane

Insertion of proteins into the membranes
Proteins that are secreted from the cell pass through
a membrane while remaining in the aqueous channel
of the translocon
By contrast, proteins that reside in membranes start
the process in the same way, but then transfer from
the aqueous channel into the hydrophobic
environment
The challenge in accounting for insertion of proteins
into membranes is to explain what distinguishes
transmembrane proteins from secreted proteins, and
causes this transfer

The pathway by which proteins of either type I or type
II are inserted into the membrane follows the same
initial route as that of secretory proteins, relying on a
signal sequence that functions co-translationally
But proteins that are to remain within the membrane
possess a second, stop-transfer signal
This takes the form of a cluster of hydrophobic amino
acids adjacent to some ionic residues
The cluster serves as an anchor that attaches on to
the membrane and stops the protein from passing
right through

Proteins that are secreted fr
while remaining in the aqu
trast, proteins that reside in me
way, but then transfer from the
environment. The challenge in a
membranes is to explain what
from secreted proteins, and caus
proteins of either type I or type
lows the same initial route as tha
nal sequence that functions co-t
remain within the membrane p
This takes the form of a cluster
some ionic residues. The cluste
the membrane and stops the pro
A surprising property of anc
as signal sequences when engi
placed into a protein lacking oth
sor membrane translocation. On
is that the signal sequence and
common component of the appa
signal sequence initiates translo
sequence displaces the signal se
The insertion of type I protein
sequence is N-terminal. The locat
transfer of the protein is halted. W
membrane, domains on the N-ter
while domains on the C-terminal
A common location for a sto
C-terminus. As shown in the fi
P r o t e i n s t h a t r e s i d e i n
membranes enter the same
route as secreted proteins, but
transfer is halted when anchor
sequence passes into the
membrane. If the anchor is at the
C-terminus, the bulk of the
protein passes through the
membrane and is exposed on far
surface.

Type II proteins do not have a cleavable leader sequence at
the N-terminus
Instead the signal sequence is combined with an anchor
sequence
Membrane insertion starts by the insertion of the signal
sequence in the form of a hairpin loop
The signal sequence enters the membrane, but the joint
signal-anchor sequence does not pass through
Instead it stays in the membrane (perhaps interacting directly
with the lipid bilayer), while the rest of the growing polypeptide
continues to loop into the endoplasmic reticulum

The signal-anchor sequence is usually internal, and its location
determines which parts of the protein remain in the cytosol and
which are extracellular
Essentially all the N-terminal sequences that precede the
signal-anchor are exposed to the cytosol
Usually the cytosolic tail is short, ~6-30 amino acids
In effect the N-terminus remains constrained while the rest of
the protein passes through the membrane
This reverses the orientation of the protein with regard to the
membrane

A combined signal-anchor
causes a protein to reverse its
orientation, so that N-terminus
remains on the inner surface
and the C-terminus is exposed
on the outer surface of the
membrane

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How multiple transmembrane protein
inserted in the lipid inlayer is not well know

Targeting protein to membrane bounded organelles
• Post-translational translocation
• Post-translational membrane insertion depends
on leader sequences
• Many proteins that enter mitochondria or chloroplasts have
leader sequences - responsible for primary recognition of
the outer membrane of the organelle
The leader sequence initiates the interaction between the
precursor and the organelle membrane
- The protein passes through the membrane
- The leader is cleaved by a protease on the organelle side

• Mitochondria and chloroplasts synthesize only some
of their proteins
• Mitochondria synthesize only ~l0 organelle proteins;
chloroplasts synthesize ~50 proteins
• The majority of organelle proteins are synthesized in
the cytosol by the same pool of free ribosomes that
synthesize cytosolic proteins
• They must then be imported into the organelle

Leader sequences
allow proteins to
r e c o g n i z e
m i t o c h o n d r i a o r
chloroplast surfaces by
a post-translational
process

• The leaders of proteins imported into mitochondria
and chloroplasts usually have both hydrophobic and
basic amino acids
• They consist of stretches of uncharged amino acids
interrupted by basic amino acids, and they lack acidic
amino acids
• Recognition of the leader does not depend on its
exact sequence
- but rather on its ability to form an amphipathic helix:
ü one face has hydrophobic amino acids, and the other
face presents the basic amino acids

The leader sequence of yeast cytochrome c oxidase subunit IV
consists of 25 neutral and basic amino acids. The first 12 amino
acids are sufficient to transport any attached polypeptide into the
mitochondrial matrix

• The leader sequence contains all the information
needed to localize an organelle protein
• The ability of a leader sequence can be tested by:
• Constructing an artificial protein - a leader from
an organelle protein is joined to a cytosolic protein
• Constructing a hybrid gene, which is then
translated into the hybrid protein
• Several leader sequences have been shown to
function independently to target any attached
sequence to the mitochondrion or chloroplast

The leader sequence and the transported protein
represent domains that fold independently
Irrespective of the sequence to which it is attached, the
leader must be able to fold into an appropriate structure
to be recognized by receptors on the organelle envelope
The attached polypeptide sequence plays no part in
recognition of the envelope
Ex.
if the leader sequence is attached to the cytosolic
protein DHFR (dihydrofolate reductase), the DHFR
becomes localized in the mitochondrion

Restrictions on transporting a hydrophilic protein through
the hydrophobic membrane:
Observation: methotrexate, a ligand for the enzyme
DHFR, blocks transport into mitochondria of DHFR
fused to a mitochondrial leader
The tight binding of methotrexate prevents the enzyme
from unfolding when it is translocated through the
membrane
→ Although the sequence of the transported protein is
irrelevant for targeting purposes,
In order to follow its leader through the membrane,
it requires the flexibility to assume an unfolded
conformation

A hierarchy of sequences determines location within
organelles
The N-terminal part of a leader sequence targets a
protein to the mitochondrial matrix or chloroplast
lumen
An adjacent sequence can control further targeting, to
a membrane or the intermembrane spaces
The sequences are cleaved successively from the
protein

The mitochondrion is surrounded by an envelope
consisting of two membranes
Proteins imported into mitochondria may be located in
the outer membrane, the intermembrane space, the
inner membrane, or the matrix
A protein that is a component of one of the
membranes may be oriented so that it faces one side
or the other
What is responsible for directing a mitochondrial
protein to the appropriate compartment?

The "default" pathway for a protein imported into a
mitochondrion is to move through both membranes into the
matrix
A protein that is localized within the intermembrane space or
in the inner membrane itself requires an additional signal,
which specifies its destination within the organelle
A multipart leader contains signals that function in a
hierarchical manner
The first part of the leader targets the protein to the organelle,
and the second part is required if its destination is elsewhere
than the matrix
The two parts of the leader are removed by successive
cleavages

M i t o c h o n d r i a h a v e
receptors for protein
transport in the outer and
i n n e r m e m b r a n e s .
Recognition at outer
membrane may lead to
transport through both
receptors into the matrix,
where the leader is
cleaved. If it has a
membrane targering signal
it may be re-exported
The mitochondrion is surrounde
membranes. Proteins imported
the outer membrane, the intermemb
the matrix. A protein that is a comp
be oriented so that it faces one side o
What is responsible for directing
propriate compartment? The "defau
into a mitochondrion is to move thr
trix. This property is conferred by t
quence. A protein that is localized
in the inner membrane itself require
ifies its destination within the org
signals that function in a hierarc
Figure 8.39. The first part of the l
ganelle, and the second part is req
than the matrix. The two parts of th
cleavages.
Cytochrome cl is an example.
and faces the intermembrane space
amino acids, and can be divided in
The sequence of the first 32 amino
half of this region, can transport DH
the first part of the leader sequence
prises a matrix-targeting signal. Bu
tached sequence—such as murine

The leader of yeast cytochrome c1 contains an N-terminal
region that targets the protein to the mitochondrion,
followed by a region that targets the (cleaved) protein to
the inner membrane. The leader is removed by two
cleavage events.
tease is involve
the final destin
Mg2+-dependen
sequence mus
reside in the in

If
v-
er
i-
ne
he
h
e-
w-
er
ne
ar
st
Chloroplast targeting
A protein approaches the
chloroplast from the cytosol
with about 50 residue
leader. The N-terminal half
of the leader sponsors
passage into the envelope
or through it into the
stroma. Cleavage occurs
during envelope passage.

Chloroplast targeting
Passage through chloroplast membranes is achieved
in a similar manner.
They pass the outer and inner membranes of the
envelope into the stroma, a process involving the
same types of passage as into the mitochondrial
matrix
But some proteins are transported yet further, across
the stacks of the thylakoid membrane into the lumen.
Proteins destined for the thylakoid membrane or
lumen must cross the stroma en route

Chloroplast targeting signals resemble mitochondrial
targeting signals
The leader consists of ~50 amino acids, and the N-
terminal half is needed to recognize the chloroplast
envelope
A cleavage between positions 20-25 occurs during or
following passage across the envelope, and proteins
destined for the thylakoid membrane or lumen have a
new N-terminal leader that guides recognition of the
thylakoid membrane
There are several (at least four) different systems in
the chloroplast that catalyze import of proteins into the

The general principle governing protein transport into
mitochondria and chloroplasts therefore is that:
• The N-terminal part of the leader targets a protein
to the organelle matrix, and an additional sequence
(within the leader) is needed to localize the protein
at the outer membrane, intermembrane space, or
inner membrane.
The additional sequence may function when it
becomes N-terminal, after the first cleavage event

There are different receptors for transport through
each membrane in the chloroplast and mitochondrion
In the chloroplast they are called TOC and TIC, and
in the mitochondrion they are called TOM and TIM,
referring to the outer and inner membranes,
respectively

The TOM complex consists of ~9 proteins, many of
The TOM aggregate has a size of > 5 00 kD, with a
diameter of ~138 A, and forms an ion-conducting
channel.
A complex contains 2-3 individual rings of diameter
75 A, each with a pore of diameter 20 A.

The
gral m
Figure
ter of
tains
diamet
To
channe
the ou
which
are tw
subcom
are im
subcom
minal
recept
TOM proteins form receptor complexes that are needed
for translocation across the mitochondrion outer
membrane

Tom40 is deeply imbedded in the membrane and
provides the channel for translocation. It contacts
preproteins as they pass through the outer
membrane
It binds to three smaller proteins, Tom5,6,7, which
may be components of the channel or assembly
factors
There are two subcomplexes that provide surface
receptors
Tom20,22 form a subcomplex with exposed domains
in the cytosol

Most proteins that are imported into mitochondria are
recognized by the Tom20,22 subcomplex, which is
the primary receptor and recognizes the N-terminal
sequence of the translocating protein
Tom37,70,71 provides a receptor for a smaller
number of proteins that have internal targeting
sequences.

When a protein is translocated through the TOM complex, it
passes from a state in which it is exposed to the cytosol into a
state in which it is exposed to the intermembrane space.
However, it is not usually released, but instead is transferred
directly to the TIM complex.
It is possible to trap intermediates in which the leader is
cleaved by the matrix protease, while a major part of the
precursor remains exposed on the cytosolic surface of the
envelope
This suggests that a protein spans the two membranes during
passage.
The TOM and TIM complexes do not appear to interact directly
(or at least do not form a detectable stable complex), and they
may therefore be linked simply by a protein in transit.

When a translocating protein reaches the
intermembrane space, the exposed residues may
immediately bind to a TIM complex, while the rest of
the protein continues to translocate through the TOM
complex.
There are two TIM complexes in the inner membrane.
The Tim 17-23 complex translocates proteins to the
lumen
Substrates are recognized by their possession of a
positively charged N-terminal signal
Tim 17-23 are transmembrane proteins that comprise
the channel

Figure 8.43 shows that they are associated with
Tim44 on the matrix side of the membrane. Tim 44 in
turn binds the chaperone Hsp70
This is also associated with another chaperone, Mge,
the counterpart to bacterial GrpE
This association ensures that when the imported
protein reaches the matrix, it is bound by the Hsp70
chaperone
The high affinity of Hsp70 for the unfolded
conformation of the protein as it emerges from the
inner membrane helps to "pull" the protein through the
channel.

seque
W
from
is ex
lease
sible
prote
tosol
two m
appe
comp
sit. W
expo
rest o
T
T
strate
mina
chan
Tim proteins form receptor complexes that are needed for
translocation across the mitochondrion outer membrane

A major chaperone activity in the mitochondrial matrix
is provided by Hsp60 (which forms the same sort of
structure as its counterpart GroEL)
Association with Hsp60 is necessary for joining of the
subunits of imported proteins that form oligomeric
complexes.
An imported protein may be "passed on" from Hsp70
to Hsp60 in the process of acquiring its proper
conformation.
The Tim22-54 complex translocates proteins that
reside in the inner membrane.

How does a translocating protein finds its way from
the TOM complex to the appropriate TIM complex?
Two protein complexes in the intermembrane space
escort a translocating protein from TIM to TOM
The Tim9-10 and Tim8-13 complexes act as escorts
for different sets of substrate proteins
Tim9-10 may direct its substrates to either Tim22-54
or Tim23-17, while Tim8-13 directs substrates only to
Tim22-54
Some substrates do not use either Tim9-10 or
Tim8-13, so other pathways must also exist. The
pathways are summarized in Figure 8.44

channel. Figure 8.43 sho
matrix side of the mem
Hsp70. This is also asso
terpart to bacterial GrpE
ported protein reaches th
The high affinity of Hsp7
as it emerges from the
through the channel.
A major chaperone a
by Hsp60 (which forms
GroEL). Association wi
units of imported prote
ported protein may be "p
of acquiring its proper c
The Tim22-54 compl
membrane.
How does a transloca
plex to the appropriate T
termembrane space esco
The Tim9-10 and Tim8-1
substrate proteins. Tim9
or Tim23-17, while Tim8
substrates do not use e
Tim9-10 takes proteins
form TOM to either TIM
complex and Tim9-13
takes proteins to Tim
22-54.

What is the role of the escorting complexes? They
may be needed to help the protein exit from the TOM
complex as well as for recognizing he TIM complex.
The Figure below shows that a translocating protein
may pass directly from the TOM channel to the
Tim9,10 complex, and then into the Tim22-54 channel

A mitochondrial protein folds under different
conditions before and after its passage through the
membrane
Ionic conditions and the chaperones that are present
are different in the cytosol and in the mitochondrial
matrix
It is possible that a mitochondrial protein can attain its
mature conformation only in the mitochondrion.

A translocating
protein may be
transferred directly
from TOM to
Tim22-44

Peroxisomes translocation system
• Proteins are imported into peroxisomes in their fully
folded state
• They have either a PTS1 sequence at the C-
terminus or a PTS2 sequence at the N-terminus
• The receptor Pex5p binds the PTS1 sequence, and
the receptor Pex7p binds the PTS2 sequence
• The receptors are cytosolic proteins that shuttle into
the peroxisome carrying a substrate protein and
then return to the cytosol

Several peroxisomal proteins are
teins from the cytosol. The peroxiso
types of signals are called Pex5p and P
teins are part of membrane-associated
translocation reaction.
Transport into the peroxisome has
tant differences from the system used f
Proteins can be imported into the p
folded state. This contrasts with the
for passage into the ER or mitochond
channel in the membrane into the or
unfolded thread of amino acids. It is
preexisting channel could expand to p
resurrect an old idea and to suppose th
the substrate protein when it associate
The Pex5p and Pex7p receptors are
but are largely cytosolic, with only a
peroxisomes. They behave in the same
isome and the cytosol. Figure 8.46 sho
strate protein in the cytosol, takes it t
through the membrane into the interio
to undertake another cycle. This shut
The Pex5p receptor binds a
substrate protein in the
cytosol, carries it across the
m e m b r a n e i n t o t h e
peroxisome and returns to
the cytosol
The receptor binds a substrate
protein in the cytosol, takes it to
the peroxisome, moves with it
through the membrane into the
interior, and then returns to the
cytosol to undertake another
cycle
T h i s s h u t t l i n g b e h a v i o r
resembles the carrier system for
import into the nucleus.

Peroxisomes are small bodies (05-1.5 µm diameter)
enclosed by a single membrane
They contain enzymes concerned with oxygen
utilization, which convert oxygen to hydrogen peroxide
by removing hydrogen atoms from substrates
Catalase then uses the hydrogen peroxide to oxidize a
variety of other substrates
Their activities are crucial for the cell , since the fatal
disease of Zellweger syndrome was found to be
caused by lack of peroxisomes, > 15 human diseases
have been linked to disorders in peroxisome function

Transport of proteins to peroxisomes occurs post-
translationally
Proteins that are imported into the matrix have either
of two short sequences, called PTS1 and PTS2
The PTS1 signal is a tri- or tetrapeptide at the C-
terminus
It was originally characterized as the sequence SKL
(Ser-Lys-Leu), but now a large variety of sequences
have been shown to act as a PTS1 signal

The addition of a suitable sequence to the C-terminus
of cytosolic proteins is sufficient to ensure their import
into the organelle
The PTS2 signal is a sequence of 9 amino acids,
again with much diversity, and this can be located
near the N-terminus or internally
Several peroxisomal proteins are necessary for the
import of proteins from the cytosol
.

The peroxisomal receptors that bind the two types of
signals are called Pex5p and Pex7p, respectively
The other proteins are part of membrane-associated
complexes concerned with the translocation reaction
Transport into the peroxisome has unusual features
that mark important differences from the system used
for transport into other organelles
Proteins can be imported into the peroxisome in their
mature, fully folded state

This contrasts with the requirement to unfold a protein
for passage into the ER or mitochondrion, where it
passes through a channel in the membrane into the
organelle in something akin to an unfolded thread of
amino acids
It is not clear how the structure of a preexisting
channel could expand to permit this
One possibility is to resurrect an old idea and to
suppose that the channel assembles around the
substrate protein when it associates with the
membrane

The Pex5p and Pex7p receptors are not integral
membrane proteins, but are largely cytosolic, with only
a small proportion associated with peroxisomes
They behave in the same way, cycling between the
peroxisome and the cytosol
.

Pores are used for nuclear import and export
The space between the inner & outer nuclear membranes is
continuous with the lumen of the endoplasmic reticulum
The two membranes come into contact at openings called nuclear
pore complexes
At the center of each complex is a pore that provides a water-
soluble channel between nucleus and cytoplasm
→ the nucleus and cytosol have the same ionic milieu
There are ∼3000 pore complexes on the nuclear envelope of
an animal cell
The nuclear pores are used for both import and export of
material

Nuclear pores are used for import and export

Transport between nucleus and cytoplasm proceeds in
both directions
Since all proteins are synthesized in the cytosol any
proteins required in the nucleus must be transported
there
Since all RNA is synthesized in the nucleus, the entire
cytoplasmic complement of RNA (mRNA, rRNA, tRNA,
and other small RNAs) must be derived by export from
the nucleus
The nuclear pores are used for both import and export
of material

We can form an impression of the magnitude of import
by considering the histones, the major protein
components of chromatin
In a dividing cell, enough histones must be imported
into the nucleus during the period of DNA synthesis to
associate with a diploid complement of chromosomes
Since histones form about half the protein mass of
chromatin, we may conclude that overall about 200
chromosomal protein molecules must be imported
through each pore per minute.

Uncertainties about the processing and stability of mRNA make
it more difficult to calculate the number of mRNA molecules
exported, but to account for the ~250,000 molecules of mRNA
per cell probably requires ~1 event per pore per minute
The major RNA synthetic activity of the nucleus is of course the
production of rRNA, which is exported in the form of assembled
ribosomal subunits
Just to double the number of ribosomes during one cell cycle
would require the export of ~5 ribosomal subunits (60S and
40S) through each pore per minute
.

For ribosomal proteins to assemble with the rRNA, they must
first be imported into the nucleus
So ribosomal proteins must shuttle into the nucleus as free
proteins and out again as assembled ribosomal subunits
• Given ~80 proteins per ribosome, their import must be
comparable in magnitude to that of the chromosomal
proteins

Nuclear pores are large symmetrical structures
• Nuclear pore complexes have a uniform appearance
when examined by microscopy
• The pores can be released from the nuclear
envelope by detergent
• They appear as annular structures, consisting of
rosettes made of 8 spokes

A model for the nuclear pore shows 8-
fold symmetry. Two rings from the
upper and lower surfaces (shown in
yellow); they are connected by the
spokes (shown in green on the inside
and blue on the outside).
Nuclear pores appear as annular
structures by electron microscopy. The
circle around one pore has a diameter
of 120 nm.

The 8 radial arms outside the ring may be responsible
for anchoring the pore complex in the nuclear
envelope; they penetrate the membrane
The 8 interior spokes project from the ring, closing the
opening to a diameter of ~48 nm
Within this region is the transporter, which contains a
pore that approximates a cylinder <10 nm in diameter

The outsides of the nuclear
coaxial (cytoplasmic and
nucleoplasmic) rings are
connected to radial arms. The
interior is connected to spokes
that project towards the
transporter that contains the
central pore.

Proteins require signals to be transported through
the pore
The nuclear localization signal (NLS) - the most
common motif responsible for import into the nucleus
Mutation of the signal can prevent the protein from
entering the nucleus
Many NLS sequences take the form of a short, basic
stretch of amino acids
Often there is a proline residue to break α-helix formation
upstream of the basic residues
Hydrophobic residues are rare
Some NLS signals are bipartite and require two separate
short clusters

Nuclear localization signals have basic residues.
NLS sequences are interchangeable - they are all
recognized by the same import system

Transport receptors carry cargo proteins through the
pore
A carrier protein (or transport receptor) takes the
substrate through the pore
The transport receptor must then be returned across the
membrane to function in another cycle
The transport receptors are classified according to the
direction in which they transport the cargo
Importins - bind the cargo in the cytoplasm and release
it in the nucleus
Exportins - bind the cargo in the nucleus and release it
in the cytoplasm

A carrier protein binds to a substrate, moves with it through the nuclear pore,
is released on the other side, and must be returned to reuse.

Bacterial translocation system
Bacterial proteins that are exported to or through
membranes use both post-translational and co-
translational mechanisms
The bacterial envelope consists of two membrane
layers
The space between them is called the periplasm
Proteins are exported from the cytoplasm to reside in
the envelope or to be secreted from the cell

The mechanisms of secretion from bacteria are similar
to those characterized for eukaryotic cells,
Proteins that are exported from the cytoplasm have
one of four fates:
Proteins are to be:
1. Inserted into the inner membrane
2. Translocated through the inner membrane to rest in
the periplasm
3. Inserted into the outer membrane
4. Translocated through the outer membrane into the
medium

8.21 Bacteria u
post-translatio
Key Concepts
• Bacterial proteins t
both post-translatio
The bacterial enve
between them is
the cytoplasm to resid
The mechanisms of se
terized for eukaryotic
nents. Figure 8.47 s
cytoplasm have one o
Bacterial proteins may
be exported either post-
translationally or co-
translationally and may
be located within either
m e m b r a n e o r t h e
periplasmic space or
may secreted

The Sec system transports proteins into and through
the inner membrane
The bacterial SecYEG translocon in the inner
membrane is related to the eukaryotic Sec61
translocon
Various chaperones are involved in directing secreted
proteins to the translocon.

There are several systems for transport through the
inner membrane
The best characterized is the Sec system, whose
components are shown in Figure below
The translocon that is embedded in the membrane
consists of three subunits that are related to the
components of mammalian/yeast Sec61.
Each of the subunits is an integral transmembrane
protein.

(SecY has 10 transmembrane segments and SecE
has 3 transmembrane segments.
The functional translocon is a trimer with one copy of
each subunit
The major pathway for directing proteins to the
translocon consists of SecB and SecA
SecB is a chaperone that binds to the nascent protein
to control its folding
It transfers the protein to SecA, which in turn
transfers it to the translocon.

ible
pass
tion
and
end-
nser-
into
nsla-
ost-
tion
with
ader
sev-
rane
ead-
nes,
atus.
ding
uta-
The Sec system has the SecYEG translocon embedded in the
membrane, the SecA associated protein that pushes protein
through the channel, SecB chaperone that transfers the
nascent proteins to SecA and the signal peptidase that
cleaves the N-terminal signal from the translocated protein.

There are two predominant ways of directing proteins
to the Sec channel (Figure below):
• The SecB chaperone;
• and the 4.5S RNA-based SRP.

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file again. If the red x still appears, you may have to delete the image and then insert it again.
SecB/SeB transfer
p r o t e i n s t o t h e
translocon in order for
them to pass through
the membrane. 4.5S
RNA transfer proteins
t h a t e n t e r t h e
membrane

The SecB transfers the nascent
protein to SecA which inserts to
the channel. Translocation
requires the hydrolysis of ATP
and promotive force. SecA
u n d e r g o e s c y c l e s o f
associationa and dessociation
with the channel and provides
the motive force to push the
protein through
newly synthesized protein. Second, it has an
SecA. This allows it to target a precursor prote
SecB-SecYEG pathway is used for translocatio
creted into the periplasm and is summarized in
SecA is a large peripheral membrane pro
ways to associate with the membrane. As a pe
tein, it associates with the membrane by virtu
lipids and for the SecY component of the trans
a multisubunit complex that provides the tra
ever, in the presence of other proteins (SecD
found as a membrane-spanning protein. It prob
that pushes the substrate protein through the S
SecA recognizes both SecB and the precurs
ones; probably features of the mature protein seq
are required for recognition. SecA has an ATP
upon binding to lipids, SecY, and a precursor p
tions in a cyclical manner during translocation. A
sor protein, it binds ATP, and -20 amino acids ar
membrane. Hydrolysis of ATP is required to re
SecA. Then the cycle may be repeated. Precursor
provide the spur to bind more ATP, translocate a
and release the precursor. SecA may alternate b
integral membrane forms during translocation; w
domain of SecA may insert into the membrane a
Another process can also undertake translo
is released by SecA, it can be driven through
tonmotive force (that is, an electrical potentia

Summary
• Passage across a membrane requires a special
apparatus
• Protein translocation may be post-translational or
co-translational
• Chaperones may be required for protein folding
• Signal sequences initiate translocation
• The signal sequence interacts with the SRP
• The SRP interacts with the SRP receptor
• Translocation requires insertion into the translocon
and (sometimes) a ratchet in the ER

Summary
• Reverse translocation sends proteins to the
cytosol for degradation
• Proteins reside in the membrane by means of
hydrophobic regions
• Anchor sequences determine protein orientation
• Transfer of transmembrane domains from the
translocon into the lipid bilayer is triggered by the
interaction of the transmembrane region with the
translocon.
• Post-translational membrane insertion depends on
leader sequences

Summary
• A hierarchy of sequences determines location
within organelles
• Inner and outer mitochondrial membranes have
different translocons
• Peroxisomes employ another type of translocation
system
• Bacteria use both co-translational and post-
translational translocation
• The Sec system transports proteins into and
through the inner membrane
• Pores are used for nuclear import and export

Summary
• Nuclear pores are large symmetrical structures
• The nuclear pore is a size-dependent sieve for
smaller material
• Proteins require signals to be transported through
the pore
• Transport receptors carry cargo proteins through
the pore
• Ran controls the direction of transport

Proteins enter the pathway that leads to secretion by co-
translational transfer to the membranes of the ER
They are then transferred to the Golgi apparatus, where they
are sorted according to their final intended destination
Proteins are carried forward or diverted to other organelles
Their destinations are determined by specific sorting signals,
which take the form of short sequences of amino acids or
covalent modifications that are made to the protein
Protein trafficking

Key concepts
• Translocation: Ribosomes --- ER
• Signal/leader sequences – specific addresses
• Ribosomes---ER---Golgi apparatus---Plasma membrane---
Extracellular---Exocytosis
• Endocytosis
• Trafficking —Glycosylation—oligosaccharide
• Specific signals (sorting signals)-specific addresses
1. N-acetyl glucosamine
2. mannose, and
3. glucose residues is formed on a special lipid, dolichol
• Vesicles
• Coated proteins (COP I and COP II)
Protein trafficking

The transport machinery consists of small membranous
vesicles
A vesicle buds off from a donor surface and then fuses with a
target surface
Proteins are released into the lumen or into the membrane of
the target compartment, depending on their nature, and must
be loaded into new vesicles for transport to the next
compartment
The series of events is repeated at each transition between
membrane surfaces, for example, during passage from the ER
to the Golgi, or between cisternae of the Golgi stacks

Two important changes occur to a protein in the ER:
• It becomes folded into its proper conformation; and
• Modified by glycosylation
A protein is translocated into the ER in unfolded form
• Folding occurs as the protein enters the lumen

Multimeric glycoproteins usually oligomerize in the
ER
Oligomers are rapidly transported from the ER to the
Golgi, but unassembled subunits or misassembled
proteins are held back
Misfolded proteins are often associated with ER-
specific chaperones and removed by degradation
So a protein is allowed to move forward into the Golgi
only if it has been properly folded previously in the
ER

Oligosaccharides are added to proteins in the ER and
Golgi---Glycosylation in a specific order
The Golgi stacks are polarized: Cis and Trans
Coated vesicles transport both exported and imported
proteins
Different types of coated vesicles exist in each
Pathway
Vesicles can bud and fuse with membranes
The exocyst tethers vesicles by interacting with rab/
chaperone

Specific signals (sorting signals) cause proteins to be:
• Returned from the Golgi to the ER
• Retained in the Golgi
• Retained in the plasma membrane, or
• Transported to endosomes and lysosomes
• Proteins may be transported between the plasma
membrane and endosomes

• Vesicles are released when they bud from a donor
compartment and are surrounded by coat proteins
• During fusion, the coated vesicle binds to a target
compartment, is uncoated, and fuses with the
target membrane, releasing its contents

Oligosaccharides are added to proteins in the ER and
Golgi
• A major function of the ER and Golgi is to glycosylate
proteins as they pass through the system
• N-linked oligosaccharides are initiated by transferring
the saccharide from the lipid dolichol to an
asparagine of the target protein in the ER
• Sugars are trimmed in the ER to give a high
mannose oligosaccharide
• A complex oligosaccharide is generated in those
cases in which further residues are added in the
Golgi

Virtually all proteins that pass through the secretory apparatus
are glycosylated
Glycoproteins are generated by the addition of oligosaccharide
groups either to:
• The NH2 group of asparagine (N-linked glycosylation) or
• The OH group of serine, threonine, or hydroxylysine (O-linked
glycosylation)
N-linked glycosylation is initiated in the endoplasmic reticulum
and completed in the Golgi
O-linked glycosylation occurs in the Golgi alone

An oligosaccharide containing:
• 2 N-acetyl glucosamine
• 9 mannose, and
• 3 glucose residues is formed on a special lipid, dolichol
Dolichol is a highly hydrophobic lipid that resides within the ER
membrane, with its active group facing the lumen
The oligosaccharide is constructed by adding sugar residues
individually
It is linked to dolichol by a pyrophosphate group, and is
transferred as a unit to a target protein by a membrane-bound
glycosyl transferase enzyme whose active site is exposed in the
lumen of the endoplasmic reticulum

Dolichol refers to any of a group of long-chain mostly
unsaturated organic compounds that are made up of varying
numbers of isoprene units terminating in an α-saturated
isoprenoid group, containing an alcohol functional group

Structure of Dolichol and their function during protein N-glycosylation. (A)
Structure of Dolichol, where n is dependent on particular cis-
prenyltransferase. Dolichol phosphate is an isoprenoid compound (90-100
carbons total) made from dolichol by phosphorylation. Dolichol phosphate
performs important functions in synthesis of N-linked glycoproteins, as
illustrated in (B). Dolichol phosphate is the structure upon which the complex
oligosaccharide is made before transfer to the target protein. The addition of
the first moiety, N-acetylglucosamine from UDP-N-acetylglucosamine, can
be blocked by the antibiotic, tunicamycin. After assembly of the
oligosaccharide is complete, the carbohydrate structure is transferred from
dolichol phosphate to an asparagine residue of a target protein having the
sequence Asn-x-Ser/Thr, where × is any amino acid. As also shown in (B),
Dolichol phosphate can also act as a carrier of sugars to oligosaccharide
chain synthesis assembly; such activated sugars include dolichol-P-
mannose and dolichol-P-glucose.

N-Acetylglucosamine is an amide derivative of the
monosaccharide glucose. It is a secondary amide between
glucosamine and acetic acid. It is significant in several
biological systems.

Mannose is a simple hexose sugar that occurs naturally in
some plants, including cranberries

the necessary functions all
cates through the membra
folding and oligomerizati
needed to catalyze the pro
enough in the cell.
Multimeric glycoprot
oligomerization may be n
rapidly transported from t
or misassembled proteins
associated with ER-spec
removed by degradation.
the Golgi only if it has be
27.2 Oligosaccha
the ER and Golgi
Key Concepts
• A major function of the
they pass through the
• N-linked oligosaccharide
from the lipid dolichol to
• Sugars are trimmed in
oligosaccharide.
An oligosaccharide is
formed from dolichol and
transferred by glyscosyl
transferase to asparagine
of a target protein

The acceptor group is an asparagine residue, located within the
sequence Asn-X-Ser or Asn-X-Thr (where X is any amino acid
except proline)
It is recognized as soon as the target sequence is exposed in the
lumen, when the nascent protein crosses the ER membrane
Some trimming of the oligosaccharide occurs in the ER, after
which a nascent glycoprotein is handed over to the Golgi
The oligosaccharide structures generated during transport
through the ER and Golgi fall into two classes, determined by the
fate of the mannose residues:
.

High mannose oligosaccharides are generated by trimming the
sugar residues in the ER
Almost immediately following addition of the oligosaccharide, the
3 glucose residues are removed by the enzymes glucosidases I
and II
For proteins that reside in the ER, a mannosidase removes some
of the mannose residues to generate the final structure of the
oligosaccharide
The ER mannosidase attacks the first mannose quickly, and the
next 3 more slowly; the total number of mannose residues that is
removed varies with the individual substrate protein

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appears, you may have to delete the image and then insert it again.
Sugars are removed in
the ER in a fixed order,
initially comprising 3
glucoses and 1-4
mannose residues.
T h i s t r i m m i n g
g e n e r a t e s a h i g h
m a n n o s e
oligosaccharide

Processing of a
c o m p l e x
o l i g o s a c c h a r i d e
occurs in the Golgi
and trims the original
preformed unit to the
inner core

Complex oligosaccharides result from additional trimming and
further additions carried out in the Golgi
Golgi modifications occur in the fixed order
The first step is further trimming of mannose residues by Golgi
mannosidase I
Then a single sugar residue is added by the enzyme N-acetyl-
glucosamine transferase
Then Golgi mannosidase II removes further mannose residues.
This generates a structure called the inner core, consisting of the
sequence NAc-Glc-NAc-Glc-Man3
Additions to the inner core generate the terminal region

The residues that can be added to a complex oligosaccharide
include N-acetyl-glucosamine, galactose, and sialic acid (N-
acetyl-neuraminic acid)
The pathway for processing and glycosylation is highly ordered,
and the two types of reaction are interspersed in it
Addition of one sugar residue may be needed for removal of
another, as in the example of the addition of N-acetyl-
glucosamine before the final mannose residues are removed

It is not known what determines how each protein undergoes its
specific pattern of processing and glycosylation
It is assumed that the necessary information resides in the
structure of the polypeptide chain; it cannot lie in the
oligosaccharide, since all proteins subject to N-linked
glycosylation start the pathway by addition of the same
(preformed) oligosaccharide
At this point, the oligosaccharide becomes resistant to
degradation by the enzyme endoglycosidase H (Endo H)
Susceptibility to EndoH is therefore used as an operational test to
determine when a glycoprotein has left the ER

Golgi
The individual cisternae of the Golgi are organized into a series
of stacks, somewhat resembling a pile of plates
A typical stack consists of 4-8 flattened cisternae
A major feature of the Golgi apparatus is its polarity
The cis side faces the endoplasmic reticulum; the trans side in a
secretory cell faces the plasma membrane
The Golgi consists of compartments, which are named cis,
medial, trans, and TGN (trans-Golgi network), proceeding from
the cis to the trans face

Proteins enter a Golgi stack at the cis face and are modified
during their transport through the successive cisternae of the
stack
When they reach the trans face, they are directed to their
destination
Nascent proteins encounter the modifying enzymes as they are
transported through the Golgi stack
This may be partly determined by the fact that the modification
introduced by one enzyme is needed to provide the substrate for
the next, and partly by the availability of the enzymes proceeding
through the cisternae

The
ties of
signifi
these e
play a
that ar
promo
the add
Key C

The addition of a complex oligosaccharide can change the
properties of a protein significantly
Glycoproteins often have a mass with a significant proportion of
oligosaccharide. What is the significance of these extensive
glycosylations?
In some cases, the saccharide moieties play a structural role, for
example, in the behavior of surface proteins that are involved in
cell adhesion.
Another possible role could be in promoting folding into a
particular conformation
One modification— the addition of mannose-6-phosphate—
confers a targeting signal

ER-Golgi
Secreted and transmembrane proteins start on the route to
localization when they are translocated into the endoplasmic
reticulum during synthesis
Transport from the ER, through the Golgi, to the plasma
membrane occurs in vesicles
A protein is incorporated into a vesicle at one membrane surface,
and is released from the vesicle at the next membrane surface
Progress through the system requires a series of such transport
events
A protein changes its state of glycosylation as it passes through
the Golgi from cis to trans compartments

Vesicles are used to transport proteins both out of the cell and
into the cell
The secretion of proteins is called exocytosis; internalization of
proteins is called endocytosis
The cycle for each type of vesicle is similar, whether they are
involved in export or import of proteins:
• Budding from the donor membrane is succeeded by fusion with
the target membrane

Proteins are transported in coated vesicles. Constitutive (bulk
flow) transport from ER through the Golgi takes place by COP-
coated vesicles. Clathrin-coated vesicles are used for both
regulated exocytosis and endocytosis

Vesicles involved in transporting proteins have a protein layer
surrounding their membranes, and for this reason are called
coated vesicles
Different types of vesicles are distinguished by the protein coats
The coat serves two purposes:
• It is involved with the processes of budding and fusion; and
• it enables the type of vesicle to be identified, so that it is
directed to the appropriate target membrane
The coat may also play a role in the selection of proteins to be
transported

The process of generating a vesicle requires a membrane bilayer
to protrude a vesicle that eventually pinches off as a bud
Proteins concerned with this process are required specifically for
budding, and become part of the coat
In the reverse reaction, fusion is a property of membrane
surfaces
In order to fuse with a target membrane, a vesicle must be
"uncoated" by removal of the protein layer
A coated vesicle recognizes its destination by a reaction between
a protein in the vesicle membrane and a receptor in the target
membrane

A vesicle therefore follows a cycle in which it gains its coat, is
released from a donor membrane, moves to the next membrane,
becomes uncoated, and fuses with the target membrane
When a vesicle is generated, it carries proteins that were resident
in (or associated with) the stretch of membrane that was pinched
off
The interior of the vesicle has the constitution of the lumen of the
organelle from which it was generated
When the vesicle fuses with its target membrane, its components
become part of that membrane or the lumen of the compartment

Proteins that are transported by vesicles (that is, which are not
part of the structure of the vesicle itself) are called the cargo
Proteins must be sorted at each stage, when either they remain
in the compartment or are incorporated into new vesicles and
transported farther along the system

Coated vesicles transport both exported and imported proteins
• Protein transport through the ER-Golgi system occurs in
coated vesicles
• Secreted proteins move in the forward, anterograde
direction toward the plasma membrane
• There is also movement within the system in the reverse,
retrograde direction
• Proteins that are imported through the plasma membrane
also are incorporated in coated vesicles
• Coated vesicles bud from a donor membrane surface and
fuse with a target membrane surface

becomes uncoated
is generated, it car
the stretch of mem
has the constitutio
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become part of th
teins that are tran
structure of the v
sorted at each stag
incorporated into
The dynamic s
dilemma. There i
from the ER and th
forward or anterog
to pass through th
surface of the ER
the plasma memb
the Golgi apparatu
both are stable in s
brane must remain
The need to m
suggests that there
the Golgi to ER, s
this direction is ca
balance of forward
cles engaged in r
returning compone
flow might occur
such as tubules, w
Vesicle formation
results when coat
proteins bind to a
membrane, deform
it, and ultimately
s u r r o u n d a
m e m b r a n e t h a t
pinch off

Coated vesicles (COPI and COPII)
Newly synthesized proteins enter the ER and may be
transported along the ER-Golgi system
This transport is undertaken by transition vesicles, and is
common to all eukaryotic cells
Two different types of transition vesicles have been identified on
the basis of their coats:
The vesicles that were originally identified in transport between
Golgi cisternae are now called COP-I-coated vesicles
(COP is an acronym for coat protein.) They are involved in
retrograde transport

It is an open question whether they also undertake anterograde
transport
• Transition vesicles that proceed from the ER to the Golgi
have a different coat, called COP-II. Their major role
appears to be forward transport
• Some proteins are constitutively secreted, moving from the
trans- Golgi to the plasma membrane
• The vesicles that undertake this process have not been
characterized biochemically

Endocytic and secretory vesicles have clathrin as the most
prominent protein in their coats, and are therefore known as
clathrin-coated vesicles
Their structure is known in some detail. The 180 kD chain of
clathrin, together with a smaller chain of 35 kD, forms a
polyhedral coat on the surface of the coated vesicle

Chaperones may be required for protein folding
Protein Folding and Degradation
Self-assembly
- some proteins are able to acquire their mature
conformation spontaneously
A test for this ability
- to denature the protein and determine whether it can
renature into the active form
A protein that can self-assemble can fold or refold into
the active state from other conformations
Protein Folding

Ex. Ribonuclease
- when the enzyme is denatured, it can renature in vitro
into the correct conformation

In some cases, protein folding is initiated before the
completion of protein synthesis on ribosomes
Other proteins undergo the major part of their folding
after release from the ribosome in either the
cytoplasm or in specific compartments such as
mitochondria or the endoplasmic reticulum
Most proteins require other proteins called
“molecular chaperones” to fold properly in vivo
Incorrectly folded proteins are generally targeted for
degradation
The accumulation of misfolded proteins is associated
with a number of human diseases

Molecular chaperones (or, chaperonins)
They are proteins that oversee the correct folding of
other proteins
Many chaperonins are called heat shock proteins
(HSPs) - their levels increase at high temperature

Chaperonins may be divided into two main classes:
- those that prevent premature folding and
- those that attempt to rectify misfolding
Mechanistically, they act to prevent incorrect folding,
rather than actively creating a correct structure

Protein folding takes place by interactions between
reactive surfaces
These surfaces consist of exposed hydrophobic side
chains
Their interactions form a hydrophobic core
Incorrect interactions may occur unless the process
is controlled
As a newly synthesized protein emerges from the
ribosome, any hydrophobic patch in the sequence is
likely to aggregate with another hydrophobic patch

Such associations are likely to occur at random and
therefore will probably not represent the desired
conformation of the protein
Hydrophobic regions of proteins are intrinsically interactive, and will aggregate with
one another when a protein is synthesized (or denatured) unless prevented

Chaperones
- Proteins that mediate correct assembly by causing a
target protein to acquire one possible conformation
instead of others
This is accomplished by:
- binding to reactive surfaces in the target protein that
are exposed during the assembly process, and
- preventing those surfaces from interacting with other
regions of the protein to form an incorrect
conformation

Chaperones bind to interactive regions of proteins as they are synthesized to prevent
random aggregation. Regions of the protein are released to interact in an orderly
manner to give the proper conformation.

An incorrect structure may be formed either by
misfolding of a single protein or by interactions with
another protein
"macromolecular crowding" - the high density of
proteins in the cytosol
Crowding can cause folding proteins to aggregate
- Chaperones can counteract this effect
So one role of chaperones may be to protect a protein
so that it can fold without being adversely affected by
the crowded conditions in the cytosol

Chaperones are needed by newly synthesized and
by denatured proteins
Chaperones play two related roles concerned with
protein structure:
• When a protein is initially synthesized, as it exits the
ribosome to enter the cytosol, it appears in an unfolded
form
Spontaneous folding then occurs as the emerging
sequence interacts with regions of the protein that were
synthesized previously
Chaperones influence the folding process by controlling
the accessibility of the reactive surfaces

• When a protein is denatured, new regions are
exposed and become able to interact
These interactions are similar to those that occur when a
protein misfolds as it is initially synthesized
They are recognized by chaperones as comprising
incorrect folds
This process is involved in recognizing a protein that has
been denatured, and either assisting renaturation or
leading to its removal by degradation

Chaperones may also be required
- to assist the formation of oligomeric structures and
- for the transport of proteins through membranes

Once the protein has passed through the membrane,
it may require another chaperone to assist with
folding to its mature conformation
During membrane passage, control (or delay) of
protein folding is an important feature
- It may be necessary to maintain a protein in an
unfolded state before it enters the membrane
- the mature protein could simply be too large to fit into
the available channel
Chaperones may prevent a protein from acquiring a
conformation that would prevent passage through the
membrane

A protein is constrained to a narrow passage as it crosses a membrane

Two major types of chaperones
1. Hsp70 system
2. Chaperonin system
They affect folding through two different types of mechanism:
1. The Hsp70 system - consists of individual proteins that
bind to, and act on, the substrates whose folding is to be
controlled
It recognizes proteins as they are synthesized or emerge from
membranes (or when they are denatured by stress)
Basically it controls the interactions between exposed reactive
regions of the protein, enabling it to fold into the correct
conformation in situ

The components of the system:
1. Hsp70, Hsp40, and GrpE
The Hsp70 and Hsp40 proteins
- bind individually to the substrate proteins
- use hydrolysis of ATP to provide the energy for
changing the structure of the substrate protein
- work in conjunction with an exchange factor that
regenerates ATP from ADP

Proteins emerge from the ribosome or from passage through a membrane in an
unfolded state that attracts chaperones to bind and protect them from
misfolding.

2. a chaperonin system - consists of a large
oligomeric assembly (represented as a cylinder)
This assembly forms a structure into which unfolded
proteins are inserted
The protected environment directs their folding
There are two types of chaperonin system:
- GroEL/GroES (Hsp60/Hsp10) - found in all classes
of organism
-TRiC - found in eukaryotic cytosol

A chaperonin forms a large oligomeric complex and folds a
substrate protein within its interior.
•
Hs
su
jun
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The Hsp70 system and the chaperonin systems both act
on many different substrate proteins
Hsp90 protein - another system, functions in
conjunction with Hsp70
- but is directed against specific classes of proteins
that are involved in signal transduction, especially the
steroid hormone receptors and signaling kinases
Its basic function is to maintain its targets in an
appropriate conformation until they are stabilized by
interacting with other components of the pathway

Chaperone families have eukaryotic and bacterial counterparts (named in
parentheses).

Chaperonins Act by Two General Mechanisms

Chaperonins Act by Two General Mechanisms
A) Chaperonins of the Hsp70 type act during protein formation by binding to
hydrophobic patches of the protein. Once chaperonins are released, the protein
automatically folds. B) Large chaperonins, such as GroE, act after translation
by sequestering misfolded protein in a central cavity. Freed from the influences
of other molecules in the cytoplasm, the protein will fold correctly.

The Hsp70 family
It is ubiquitous
- found in bacteria, eukaryotic cytosol, in the
endoplasmic reticulum, and in chloroplasts and
mitochondria
Hsp70 has two domains:
- the N-terminal domain is an ATPase; and
- the C-terminal domain binds the substrate
polypeptide
When bound to ATP, Hsp70 binds and releases
substrates rapidly; when bound to ADP, the reactions are
slow
Recycling between these states is regulated by Hsp40
(DnaJ) and GrpE

Hsp40 (DnaJ) binds first to a nascent protein as it
emerges from the ribosome
Hsp40 contains a region called the J domain, which
interacts with Hsp70
Hsp70 (DnaK) binds to both Hsp40 and to the unfolded
protein
The J domain accounts for the specificity of the pairwise
interaction, and drives a particular Hsp40 to select the
appropriate partner from the Hsp70 family
The interaction of Hsp70 (DnaK) with Hsp40 (DnaJ)
stimulates the ATPase activity of Hsp70

The ADP-bound form of the complex remains associated
with the protein substrate until GrpE displaces the ADP
This causes loss of Hsp40 followed by dissociation of
Hsp70
The Hsp70 binds another ATP and the cycle can be
repeated
GrpE (or its equivalent) is found only in bacteria,
mitochondria, and chloroplasts
In other locations, the dissociation reaction is coupled to
ATP hydrolysis in a more complex way

Hsp40 binds the substrate and then Hsp70. ATP hydrolysis drives
conformational change. GrpE displaces the ADP; this causes the chaperones to
be released. Multiple cycles of association and dissociation may occur during
the folding of a substrate protein.

Protein folding is accomplished by multiple cycles of
association and dissociation
As the protein chain lengthens, Hsp70 (DnaK) may
dissociate from one binding site and then reassociate
with another
- thus releasing parts of the substrate protein to fold
correctly in an ordered manner
Finally, the intact protein is released from the ribosome,
folded into its mature conformation

Different members of the Hsp70 class function on
various types of target proteins
- Cytosolic proteins (Hsp70 and a related protein
called Hsc70) act on nascent proteins on ribosomes
- Variants in the ER: BiP or Grp78 in higher
eukaryotes; Kar2 in S. cerevisiae, or in mitochondria
or chloroplasts
- function in a rather similar manner on proteins as they
emerge into the interior of the organelle on passing
through the membrane

What feature does Hsp70 recognize in a target protein?
It binds to a linear stretch of amino acids embedded
in a hydrophobic context
This is buried in the hydrophobic core of a properly
folded, mature protein
Its exposure therefore indicates that the protein is
nascent or denatured
Motifs of this nature occur about every 40 amino acids
Binding to the motif prevents it from misaggregating with
another one

Hsp60/GroEL forms an oligomeric ring structure
Large (oligomeric) structures with hollow cavities are
often used for handling the folding or degradation of
proteins
The typical structure is a ring of many subunits,
forming a doughnut or cylinder
the target protein is in effect placed in a controlled
environment—the cavity—where it is closely
associated with the surrounding protein
This creates a high local concentration of binding
sites and supports cooperative interactions

In the case of folding, the closed environment prevents
the target protein from forming wrongful interactions with
other proteins
The energy for this processes is provided by hydrolysis
of ATP— the subunits of the ring are ATPases

A protein may be sequestered within a controlled environment for folding or
degradation
trans
pose
with
quen
lume
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bein

The Hsp60 class of chaperones forms a large apparatus
that consists of two types of subunit
Hsp60 itself (GroEL in E. coli) - forms a structure
consisting of 14 subunits that are arranged in two
heptameric rings stacked on top of each other in
inverted orientation
- the top and bottom surfaces of the double ring are the
same
The central hole is blocked at the equator of each ring by
the COOH ends of the subunits, which protrude into the
interior
So the ends of the double cylinder form symmetrical
cavities extending about halfway into each unit

This structure associates with a heptamer formed of
subunits of Hsp l0 (GroES in E. coli)
A single GroES heptamer forms a dome that associates
with one surface of the double ring
The dome sits over the central cavity, thus capping one
opening of the cylinder
The dome is hollow and in effect extends the cavity I into
the closed surface
We can distinguish the two rings of GroEL as the
proximal ring (bound to GroES) or the distal ring (not
bound to GroES)

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