protein sorting and targeting , protein generation and their distribution to various organelles and out of the cell.

1. PROTEIN SORTING AND TARGETING
BY
Rakesh H
Research Scholar
Department of Biotechnology
Sahyadri Science College,Shivamogga.
Kuvempu university.

2. Introduction:
Protein targeting
Protein targeting or protein sorting is the mechanism by which a cell
transports proteins to the appropriate positions in the cell or outside of it.

3. THE CENTRAL DOGMA
•DNA synthesis maintains the genetic
information and passes this to the next
generation
•RNA synthesis (transcription) is a
transfer of the information from the DNA
where it is stored into RNA which can be
transported and interpreted.
•Ribosomes translate the nucleotides on
the mRNA into amino acid sequences
producing a polypeptide

4. TRANSCRIPTION
• INITIATION
•ELONGATION
•TERMINATION

5. TRANSLATION
● Initiation – the assembly of a
ribosome on an mRNA molecule.
● Elongation – repeated cycles
of amino acid addition.
● Termination – the release of
the new protein chain.

6. PROTEIN TARGETING
 Both in prokaryotes and eukaryotes, newly synthesized
proteins must be delivered to a specific subcellular location or
exported from the cell for correct activity. This phenomenon is
called protein targeting.
• Protein targeting is necessary for proteins that are destined
to work
outside the cytoplasm.
• This delivery process is carried out based on information
contained in the protein itself.
• Correct sorting is crucial for the cell; errors can lead to
diseases.

7. PROTEIN TRANSLOCATION
 In 1970, Günter Blobel conducted experiments on
the translocation of proteins across membranes.
 He was awarded the 1999 Nobel Prize for his
findings. He discovered that many proteins have a
signal sequence, that is, a short amino acid
sequence at one end that functions like a postal
code for the target organelle.

8. TARGETING PATHWAYS

9. POSTTRANSLATIONAL TRANSLOCATION
 Even though most proteins are co translationally
translocated, some are translated in the cytosol
and later transported to their destination. This
occurs for proteins that go to a mitochondrion, a
chloroplast, or a peroxisome

10. CO TRANSLATIONAL TRANSLOCATION
Synthisised protein is transferred to an SRP receptor on
the endoplasmic reticulum (ER), a membrane-
enclosed organelle. There, the nascent protein is
inserted into the translocation complex

11. TARGETING SIGNALS
 Targeting signals are the pieces of information that enable the cellular
transport machinery to correctly position a protein inside or outside the
cell.
 This information is contained in the polypeptide chain or in the folded
protein.
 In the absence of targeting signals, a protein will remain in the
cytoplasm
 The continuous stretch of amino acid residues in the chain that enables
targeting are called signal peptides or targeting peptides.
 There are two types of targeting peptides.
 The presequences and
 The internal targeting peptides

12. THE PRESEQUENCES
 The presequences of the targeting peptides are
often found at the N-terminal extension .
 It is composed of between 6-136 basic and
hydrophobic amino acids.
 In case of peroxisomes the targeting sequence is
on the C-terminal extension mostly.
 signal sequences are removed from the finished
protein by specialized signal peptidases once the
sorting process has been completed

13. THE INTERNAL TARGETING PEPTIDES
 the targeting peptides are often found at the with in
polypeptide chain, not at any end .

15. PROTEINS CAN MOVE BETWEEN
COMPARTMENTS IN DIFFERENT WAYS
 Gated transport(Nucleus )
 Transmembrane
transport(Mitochondria,
Peroxisomes,)
 Vesicular transport (E.R)

16. GATED TRANSPORT
 The protein traffic
between the cytosol and
nucleus occurs between
topologically equivalent
spaces, which are in
continuity through the
nuclear pore
complexes.
 The nuclear pore
complexes function as
selective gates that
actively transport
specific
macromolecules and
macromolecular
assemblies,

17. TRANSMEMBRANE TRANSPORT
 Membrane-bound protein
translocators directly transport
specific proteins across a
membrane from the cytosol into
a space that is topologically
distinct.
 The transported protein molecule
usually must unfold to snake
through the translocator.
 The initial transport of selected
proteins from the cytosol into the
ER lumen or from the cytosol
into mitochondria.

18. VESICULAR TRANSPORT
 Proteins from the ER to the Golgi
apparatus and proteins to E.R,
for example, occurs in this way.
 transport intermediates— which
may be small, spherical transport
vesicles or larger, irregularly
shaped organelle fragments—
ferry proteins from one
compartment to another.
 The transfer of soluble recognized
by a complementary receptor in
the appropriate membrane.

19. GATED TRANSPORT

20. THE TRANSPORT OF MOLECULES BETWEEN THE
NUCLEUS AND THE CYTOSOL
 The nuclear envelope encloses the DNA and defines the nuclear
compartment.
 This envelope consists of two concentric membranes that are
penetrated by nuclear pore complexes.
 The inner and outer nuclear membranes are continuous, they maintain
distinct protein compositions.
 The inner nuclear membrane contains specific proteins that act as
binding sites for chromatin and for the protein meshwork of the nuclear
lamina that provides structural support for this membrane.
 The inner membrane is surrounded by the outer nuclear membrane,
which is continuous with the membrane of the ER. Like the membrane of
the ER the outer nuclear membrane is studded with ribosomes engaged
in protein synthesis .
 The proteins made on these ribosomes are transported into the
space between the inner and outer nuclear membranes (the
perinuclear space), which is continuous with the ER lumen. with
ribosomes engaged in protein synthesis.
 Bidirectional traffic occurs continuously between the cytosol and the
nucleus.
 The many proteins , histones, DNA and RNA polymerases, gene
regulatoryimported into the nuclear compartment from the cytosol,
 proteins, and RNA-processing proteins are selectively tRNAs and
mRNAs are synthesized in the nuclear compartment and then exported to
the cytosol

21. IMPORT AND EXPORT OF PROTEINS
TO NUCLEUS  Protein encodes a receptor
protein that is specialized for the
transport of a group of nuclear
proteins sharing structurally
similar nuclear localization
signals.
 Nuclear import receptors do not
always bind to nuclear proteins
directly. Additional adaptor
proteins are sometimes used that
bridge between the import
receptors and the nuclear
localization signals on the
proteins to be transported.
 Export -ribosomal subunits and
RNA molecules.
 For import and export requires
energy

22. TRANSMEMBRANE TRANSPORT

23. MITOCHONDRIA AND CHLOROPLASTS
 Mitochondria and chloroplasts are double-
membrane-enclosed organelles.
 They specialize in the synthesis of ATP, using
energy derived from electron transport and
oxidative phosphorylation in mitochondria and
from photosynthesis in chloroplasts.
 Both organelles contain their own DNA,
ribosomes , and other components required for
protein synthesis .
 Their growth depends mainly on the import of
proteins from the cytosol.

24. THE TRANSPORT OF PROTEINS INTO
MITOCHONDRIA AND CHLOROPLASTS

25. •Protein translocation across
mitochondrial membranes is
mediated by multi-subunit protein
complexes that function as protein
translocators.
•TOM ,TIM 23,TIM22 ,OXA
•TOM transports-mitochondrial
precursor proteins , nucleus-
encoded mitochondrial proteins.
•TIM23-proteins into the matrix
space.
•TIM22-mediates the insertion of
a subclass of inner membrane
proteins, including the carrier
protein that transports ADP, ATP,
and phosphate.
•OXA-mediates the insertion of
inner membrane proteins .

26. PROTEIN TRANSPORT INTO THE MITOCHONDRIA
Import of Mitochondrial Proteins
►Post-translational: Unfolded polypeptide chain
1. precursor proteins bind to receptor proteins of TOM
2. interacting proteins removed and unfolded polypetide is fed into
translocation channel
►Occurs contact sites joining IM and OM - TOM transports mito targeting signal across
OM and once it reaches IM targeting signal binds to TIM, opening channel complex thru
which protein enters matrix or inserts into IM

27. PROTEIN TRANSPORT INTO THE MITOCHONDRIA
Import of Mitochondrial Proteins
►Requires energy in form of ATP and H+ gradient and assitance of hsp70
-release of unfolded proteins from hsp70 requires ATP hydrolysis
-once thru TOM and bound to TIM, translocation thru TIM requires
electrochemical gradient

28. PROTEIN TRANSPORT INTO THE MITOCHONDRIA
Protein Transport into IM or IM Space Requires 2 Signal Sequences
1. Second signal =hydrophobic sequence; immediately after 1st signal sequence
2. Cleavage of N-terminal sequence unmasks 2nd signal used to translocate protein from
matrix into or across IM using OXA
3. OXA also used to transport proteins encoded in mito into IM
4. Alternative route bypasses matrix; hydrophobic signal sequence = “stop transfer”

29. CHLOROPLAST
 The preprotein for chloroplasts
may contain a stromal import
sequence or a stromal and
thylakoid targeting sequence.
The majority of preproteins are
translocated through the Toc
and Tic complexes located
within the chloroplast envelope.
 In the stroma the stromal
import sequence is cleaved off
and folding as well as intra-
chloroplast sorting to thylakoids
continues.
 Proteins targeted to the
envelope of chloroplasts
usually lack cleavable sorting
sequence.

30. TRANSLOCATION OF PROTEIN IN
CHLOROPLAST
 The vast majority of chloroplast proteins are
synthesized as precursor proteins (preproteins)
in the cytosol and are imported post-
translationally into the organelle.
 Most proteins that are destined for the
thylakoid membrane,
 Preproteins that contain a cleavable transit
peptide are recognized in a GTP-regulated
manner12 by receptorsof the outer-envelope
translocon, which is called theTOC complex.
 The preproteins cross the outer envelope
through an aqueous pore and are then
transferred to the translocon in the inner
envelope,which is called the TIC complex.
 The TOC and TIC translocons function
together during the translocation process
Completion of import requires energy,which
probably comes from the ATP-dependent
functioning of molecular chaperones in the
stroma.
 The stromal processing peptidase then
cleaves the transit sequence to produce the
mature form of the protein, which can fold into
its native form.

31. THE ENDOPLASMIC RETICULUM
 All eukaryotic cells have an endoplasmic reticulum (ER). Its membrane
typically constitutes more than half of the total membrane of an average
animal cell.
 The ER is organized into a netlike labyrinth of branching tubules and
flattened sacs extending throughout the cytosol , to interconnect.
 The ER has a central role in lipid and protein biosynthesis.
 Its membrane is the site of production of all the transmembrane proteins
and lipids for most of the cell’s organelles( the ER itself, the Golgi apparatus,
lysosomes, endosomes, secretory vesicles, and the plasma membrane).
 The ER membrane makes a major contribution to mitochondrial and
peroxisomal membranes by producing most of their lipids.
 almost all of the proteins that will be secreted to the cell exterior plus those
destined for the lumen of the ER, Golgi apparatus, or lysosomes are initially
delivered to the ER lumen.

32. TRANSLOCATION OF PROTIENS IN E.R

33. VESICULAR TRANSPORT

34. UTILIZATION OF DIFFERENT COATS IN
VESICULAR TRAFFIC

35. VESICULAR TRANSPORT FROM ER TO GC

37.  Those ER resident proteins that
escape from the ER are
returned to the ER by vesicular
transport.
 (A) The KDEL receptor present
in vesicular tubular clusters and
the Golgi apparatus, captures
the soluble ER resident proteins
and carries them in COPI-
coated transport vesicles
back to the ER. Upon binding its
ligands in this low-pH
environment, the KDEL receptor
may change conformation, so
as to facilitate its recruitment
into budding COPI-coated
vesicles.
 (B) The retrieval of ER proteins
begins in vesicular tubular
clusters and continues from all
parts of the Golgi apparatus.

38. TARGETING OF SECRETARY PROTEINS

39. THE GOLGI APPARATUS
 The Golgi apparatus is integral in
modifying, sorting, and packaging
these macromolecules for cell
secretion (exocytosis) or use within
the cell.
 Post office; it packages and labels
items(a mannose-6-phosphate label
to proteins destined for lysosomes)
which it then sends to different parts
of the cell.
 glycosylation refers to the enzymatic
process that
attaches glycans to proteins, lipids, or
other organic molecules.
 Glycosylation is a form of co-
translational and post-translational
modification

40. Five classes of glycans are produced:
 N-linked glycans attached to nitrogen of asparagine or arginine side-
chains. N-linked glycosylation requires participation of a special lipid
called dolichol phosphate.
 O-linked glycans attached to the hydroxy oxygen of serine,
threonine, tyrosine, hydroxylysine, or hydroxyproline side-chains, or
to oxygens on lipids such as ceramide
 phospho-glycans linked through the phosphate of a phospho-serine;
 C-linked glycans, a rare form of glycosylation where a sugar is added
to a carbon on a tryptophan side-chain
 Glypiation, which is the addition of a GPI anchor that links proteins to
lipids through glycan linkages.

41. PROTEIN TRAFFICKING OR SITE SPECIFIC TRANSPORT

42. SUMMARY
 Both in prokaryotes and eukaryotes, newly synthesized
proteins must be delivered to a specific subcellular
location or exported from the cell for correct activity. This
phenomenon is called protein targeting. Secretory
proteins have an N-terminal signal peptide which targets
the protein to be synthesized on the rough endoplasmic
reticulum (RER). During synthesis it is translocated
through the RER membrane into the lumen. Vesicles
then bud off from the RER and carry the protein to the
Golgi complex, where it becomes glycosylated. Other
vesicles then carry it to the plasma membrane. Fusion
of these transport vesicles with the plasma membrane
then releases the protein to the cell exterior.

43. REFERENCES
 Biochemistry, Third Edition ( David Hames & Nigel
Hooper, )
 Molecular Biology, Third Edition ( Phil Turner, Alexander
McLennan,Andy Bates & Mike White)
 Palade G (1975) Intracellular aspects of the process of
protein synthesis.Science 189, 347–358.
 Lodish, H., Berk, A., Zipursky, S.L., Matsudaira, P.,
Baltimore, D., Darnell, J., 2000, Molecular Cell Biology,
4th Ed., W.H. Freeman.
http://bcs.whfreeman.com/lodish5e/
 Lehninger principles of Biochemistry, Fourth edition ,
David L. Nelson, Michael M. Co