Protein trafficking in detail.

1. Intracellular Compartments and
Protein Sorting
1

2. Intracellular Compartments
 Eukaryotic cell is elaborately divided into functionally distinct
membrane enclosed compartments
 Intracellular membrane systems
- Provide increased membrane area
- Create enclosed compartments separate from cytosol
- Provide functionally specialized aqueous space
 Membrane enclosed organelle have a characteristic position
in the cytosol 2

3. The major intracellular compartments of an animal cell
3

4. Relative volumes occupied by the major intracellular compartments
in a liver cell (hepatocyte)
Intracellular compartment Percentage of total cell volume
Cytosol 54
Mitochondria 22
Rough ER cisternae 9
Smooth ER cisternae plus
Golgi cisternae
6
Nucleus 6
Peroxisomes 1
Lysosomes 1
Endosomes 1 4

5.  Smaller ratio of surface area to volume makes the plasma
membrane to be too small to sustain the many vital function
for which membranes are required
 Evolutionary, the compartments appear to evolve by pinching
off from specialized structures of the plasma membrane
 The internal part of these compartments is topologically
equivalent with the exterior of the cells
 Communication done through transport vesicles
5

6. 6
Topologically equivalent compartments in the secretory & endocytic
pathways in a eukaryotic cell

7. 7

8. 8

9. Four distinct families of intracellular compartments
 The nucleus & the cytosol, communicate through nuclear pore
complexes & are thus topologically continuous (although
functionally distinct)
 All organelles that function in the secretory & endocytic
pathways: the ER, Golgi apparatus, endosomes & lysosomes,
the numerous classes of transport intermediates such as
transport vesicles that move between them, and peroxisomes
 The mitochondria &
 Plastids (in plants only)
9

10. Protein targeting or protein sorting
 The process of directing each individual protein to a specific
destination
 Proteins can be targeted to the inner space of an organelle,
different intracellular membranes, the plasma membrane, or
to the exterior of the cell via secretion
 Information contained in the protein itself directs this delivery
process
 Proteins to be transported contain an amino acid sequence
which serves as a recognition signal for cellular sorting
complexes
10

11. Mechanisms of protein transport/trafficking
 Three mechanisms of transport
 Gated transport: NPC serves as a selective gate
 Transmembrane transport: involves translocases
 Vesicular transport: topologically equivalent
 Transfer involves recognition of sorting signals by
complimentary sorting receptors
11

12. 12
Protein traffic

13. Protein sorting
 Two types of sorting signals:
 Signal sequences
 Stretch of AAs & removed once sorting is completed
 5-10 hydrophobic AAs at the N-terminus (ER)
 +vely charged alternating with hydrophobic AAs (mitochondria)
 Signal patches
 Located distant one from the other & come together when
the protein folds 13

14. Sorting sequences
14

15. Some Typical Signal Sequences
15

16. 1) Gated transport
 Nuclear Envelope
16

17. The nuclear envelope
17

18. Nuclear lamina
 A protein meshwork structure, composed of two types of
lamin proteins termed A-type & B-type lamins
 Both A-type & B-type lamins belong to the type V
intermediate filaments (IFs)
 Both A-type & B-type lamins form homodimers as minimum
modules for the construction of the nuclear lamina via -
helical coiled-coil regions within a rod domain
18

19.  A/C-type lamins are inside, next to nucleoplasm; B-type
lamins are near the nuclear membrane (inner). They may
bind to integral proteins inside that membrane
 The lamins may be involved in the functional organization of
the nucleus
 Nuclear lamina confers structural support (physical strength)
to the NE & also acts as an anchoring site for chromosomes
& the cytoplasmic cytoskeleton
19

20. 20

21. Nuclear pores
 Nuclear envelope perforated by large, elaborate structure
known as nuclear pore complex (NPC)
 Molecular mass of 125 X 106 & composed of 30 different
proteins called nucleoporins (Nups)
 Active nucleus; the greater number of pore complexes
 Each pore complex contains >1 open aqueous channels that
allow diffusion of small molecules (particle larger than 10 nm
in DM or >60,000 Da are excluded, so actively transported)
21

22.  Some of the scaffold nucleoporins are structurally related to
vesicle coat protein complexes, such as clathrin & COPII
coatomer, which shape transport vesicles; & one protein is
used as a common building block in both NPCs & vesicle coats
 These similarities suggest a common evolutionary origin for
NPCs & vesicle coats
22

23. The arrangement of NPCs in the nuclear envelope
23

24. 24

25. 25
NPC components

26. Free diffusion through the nuclear pore complex
26

27. 27

28. NLS and NES
 NLS found only in nuclear proteins (sequence or patch) &
direct nuclear proteins to the nucleus
 Two types have been identified:
 SV40 type (1st found in the large T antigen of the SV40 virus):
PKKKRKV (Pro-Lys-Lys-Lys-Arg-Lys-Val); monopartite
 The bipartite type was 1st identified (Xenopus nucleoplasmin)
 KRPAATKKAGQAKKKK (2 basic, 10 spacer & at least 3 basic
residues out of 5 residues) 28

29. 29
Nuclear localization signals
Imported to the nucleus Remains in the cytosol

30. NLS and NES…
 NLS recognized by importins
 For example, importin  is a well characterized importin, can’t
recognize the specific sequence but can be assisted by importin
 which is an adaptor
 By contrast, the importin for the heterogeneous nuclear
ribonucleoproteins (hnRNPs) can recognize directly the specific
sequence in hnRNP. This importin is named "transportin”
30

31. Role of adaptor proteins in nuclear transport
31

32.  The signal for nuclear export is a leucine-rich domain which
can be recognized by a class of exportins called exportin 1 or
chromosomal region maintenance 1 (Crm1)
 LQLPPLERLTL (e.g., rev protein of HIV-1)
 Shuttling proteins have both NES & NLS (e.g., rev, NFAT,
hnRNPs)
 Many nuclear export receptors are structurally related to
nuclear import receptors &
 They are encoded by the same gene family of nuclear
transport receptors, or karyopherins
32

33. NLS and nuclear import
 Pore channel dilate from 9 nm to 26 nm in diameter
 A structure in the center function like a close fitting
diaphragm
 Molecular mechanism of gating is unclear, but protein is
transported in a fully folded conformation
33

34. NLS and nuclear import…
 NLS recognition by nuclear import receptors (importins)
marks initiation of the transport
 Receptors bind to both NLS and nucleoporins
 Nucleoporins contain FG repeats serving as a track for the
importins
 Transport is effected by binding, dissociation and rebinding
on adjacent repeat sequences
34

35.  Core FxFG repeats found in nucleoporins
 Each repeat is separated by a ‘linker’ region
Mechanism of import into nucleus
35

36. 36

37. 37

38. NLS and nuclear import
 Compartmentalization of Ran-GDP (cytosolic side) and Ran-
GTP (nuclear side) drives directional transport through NPCs
 Transport requires energy and provided by the monomeric G-
protein Ran
 Conversion between the two states is triggered by Guanine
nucleotide exchange factors (GEFs) & GTPase-activating
proteins (GAPs)
38

39. 39
The compartmentalization of Ran-GDP & Ran-GTP

40. 40
How GTP hydrolysis by Ran in the cytosol provides directionality
to nuclear transport

41. NES and Nuclear Export
 Export requires NES and exportins
 System works in the same fashion but in opposite direction
 Ran-GTP in the nucleus promotes cargo binding &
 Hydrolysis of Ran-GTP by GAP in the cytosol causes release
of the cargo and re-importing of the receptor
41

42. 42

43. 43
Examples of different types of nuclear transport signals

44. Controlling of nuclear transport
 Proteins having NLS and NES (shuttling proteins)
 Steady state localization is determined by relative rates of
import and export
 Nuclear: rate of import >rate of export
 Cytosolic: rate of export > rate of import
 Stringent control is exerted by turning on or off the NLS/NES
by phosphorylation of AAs close to the signal sequences e.g.,
NF-AT (gene regulatory protein) 44

45. The control of nuclear import during T-cell activation
45

46. Controlling of nuclear transport
 Binding of inhibitory proteins (anchor them to the
cytoskeleton, organelle or mask their NLS so that they can’t
interact with nuclear import receptors
 An appropriate stimulus releases the gene regulatory protein
from its cytosolic anchor or mask & it is then transported into
the nucleus
 E.g. steroid receptors, GRP involved in cholesterol metabolism
46

47. Controlling of nuclear transport
47

48. 48

49. 2) Transmembrane transport
 Common in mitochondria & plastids
49

50. A) Mitochondrial transport
 Have own genome but most proteins encoded by nuclear genome
 Specialize in ATP synthesis, using energy derived from electron
transport & oxidative phosphorylation in mitochondria & from
photosynthesis in chloroplasts
 Proteins transported as unfolded proteins
 Many proteins entering the matrix space contain a signal sequence
at their N-terminus & a signal peptidase rapidly removes after
import
 Other imported proteins: all outer membrane & many inner
membrane & intermembrane space proteins, have internal signal
sequences that are not removed
50

51.  The signal sequences that direct precursor proteins into the
mitochondrial matrix form an amphiphilic  helix;
 +vely charged residues cluster on one side of the helix, while
uncharged hydrophobic residues cluster on the opposite side
 Specific receptor proteins that initiate protein translocation
recognize this configuration rather than the precise AA
sequence of the signal sequence
51

52. A signal sequence for mitochondrial protein import
52

53.  The process of protein movement across membranes is called
protein translocation
 Mediated by a multi-subunit protein translocators
 Translocators contain some components that act as receptors
for mitochondrial precursor proteins, and other components
that form the translocation channels
 Translocation into mitochondria depends on signal sequences
& protein translocators
53

54. Mitochondrial transport
a) TOM complex (Translocator of the Outer Membrane)
 Function: transfers proteins across the outer membrane (OM)
& required for all nuclear encoded proteins
 Imports signal sequence into the intermembrane space &
helps to insert transmembrane proteins in the OM
 β-barrel proteins are particularly abundant in the OM
 Folding of -barrel proteins assisted by the SAM complex
54

55. b) TIM complexes: TIM 22 & TIM 23
 Function: transfer proteins across the inner membrane (IM)
 TIM 22 mediate insertion of a subclass of proteins into the IM
(the transporter that moves ADP, ATP & phosphate in & out
of mitochondria)
 TIM23 transports into the matrix space & helps to insert
transmembrane proteins into the inner membrane
55

56. c) OXA complex (cytochrome OXidase Activity):
- Inserts mitochondrial encoded proteins in the IM
- Assists insertion of imported inner membrane proteins that
are initially transported into the matrix space by the other
complexes
 Interacting proteins help to prevent precursor protein from
aggregating or folding up spontaneously
 Engagement with TOM strips off the interacting proteins
56

57. Protein translocators in the mitochondrial membranes
57

58. 58

59. Protein import in the OMM
59

60. Mitochondrial transport
 Scenarios for matrix importation
- Pass one membrane at a time
- Pass through both at once
 Transport to the IMM or IMS may require additional SS
- Cleavage of SS exposes another SS and protein can be
 Transported to matrix (if no another signal sequence)
 Inserted into the IMM, released in the IMS or traverse the
IMM once or many times 60

61. Protein import by mitochondria
61

62. 62
Protein import from the cytosol into the IMM & IMS
Mia = mitochondrial intermembrane space assembly

63. Mitochondrial transport
 Transport facilitated by chaperone proteins
 Energy is required for directional transport and comes from
ATP hydrolysis and the electrochemical H+ gradient
63

64. 64

65. 65

66. B) Transport to the ER
66

67. Transport to the ER
 The role of ER:
 Site of production of all transmembrane proteins & lipids for
most organelles, intracellular Ca2+ store
 Site at which most of the lipids of membranes of mitochondria &
peroxisomes are made
 Almost all of the proteins that will be secreted to the cell
exterior, destined for the lumen of the ER, Golgi, or lysosomes
are initially delivered to the ER lumen
 Captures two types of proteins as they are being synthesized:
transmembrane proteins & water-soluble proteins (fully
translocated to the ER lumen) 67

68. Isolation of purified rough & smooth microsomes from the ER
68

69. Transport to the ER
- Transmembrane proteins of the ER itself or others
- Water-soluble proteins released in the ER lumen
 Two types of import into the ER
 Co-translational: before complete synthesis of the polypeptide
chain
 Post-translational (Bip, SecA/Sec61 protein): the import of
proteins into mitochondria, chloroplasts, nucleus & peroxisomes
 Common pool of ribosomes are used to synthesize proteins
that stay in the cytosol or translocated to the ER
69

70. 70
Protein translocation

71. 71
Free and membrane-bound ribosomes

72. The signal hypothesis
 Signal sequences first described in 1970s in secreted proteins
translocated across the ER membrane
 ER signal sequences vary greatly in amino acid sequence, but
each has eight or more nonpolar amino acids at its center
 Signal sequence emerges from the ribosome it directs to a
translocator on the ER membrane that forms a pore in the
membrane through which the PPC is translocated
72

73.  A signal peptidase is closely associated with the translocator
& clips off the signal sequence during translation, &
 The mature protein is released into the lumen of the ER
immediately after its synthesis is completed
 The translocator is closed until the ribosome has bound, so
that the permeability barrier of the ER membrane is
maintained at all times
73

74. The signal hypothesis
74

75. The signal hypothesis
 The ER signal sequence is guided to the ER membrane by at
least two components:
 Signal-recognition particle (SRP), which cycles between the
ER membrane & the cytosol & binds to the signal sequence, &
 SRP receptor in the ER membrane
 SRP receptor is made up of two PPC & an integral membrane
protein exposed only on the cytosolic side
75

76.  How can the SRP bind specifically to so many d/t sequences?
 The answer has come from the crystal structure of the SRP
protein, which shows that the signal-sequence-binding site is
a large hydrophobic pocket lined by methionines
 Because methionines have unbranched, flexible side chains,
the pocket is sufficiently plastic to accommodate hydrophobic
signal sequences of different sequences, sizes & shapes
76

77. The signal recognition particle (SRP)
77

78.  The SRP is a rodlike structure, which wraps around the large
ribosomal subunit,
 With one end binding to the ER signal sequence as it emerges
from the ribosome as part of the newly made polypeptide
chain;
 The other end blocks the elongation factor binding site at the
interface between the large & small ribosomal subunits
 This block halts protein synthesis as soon as the signal
peptide has emerged from the ribosome
78

79.  The transient pause presumably gives the ribosome enough
time to bind to the ER membrane before completion of the
polypeptide chain, thereby ensuring that the protein is not
released into the cytosol
 This safety device may be especially important for secreted &
lysosomal hydrolases, which could wreak havoc in the
cytosol;
 Cells that secrete large amounts of hydrolases, however, take
the added precaution of having high concentrations of
hydrolase inhibitors in their cytosol
79

80.  The pause also ensures that large portions of a protein that
could fold into a compact structure are not made before
reaching the translocator in the ER membrane
 Thus, in contrast to the post-translational import of proteins
into mitochondria and chloroplasts, chaperone proteins are
not required to keep the protein unfolded
80

81. 81
The signal recognition particle (SRP)

82. 82
ER signal sequences & SRP direct ribosomes to the ER membrane

83. The signal hypothesis
 GTP hydrolysis (SRP & receptor) ensures that release occurs
after the ribosome has become properly engaged with the
translocator
 The polypeptide chain passes through an aqueous channel in the
translocator
 The translocator:
- Has a water filled pore
- Pore opens when ribosome binds
- Ribosome forms a tight seal (space continuous)
- Signal peptide binding removes a luminal protein from the pore
83

84.  The core of the translocator, called the Sec61 complex, is built
from three subunits that are highly conserved from bacteria to
eukaryotic cells
 The structure of the Sec61 complex suggests that  helices
contributed by the largest subunit surround a central channel
through which the polypeptide chain traverses the membrane
 The channel is gated by a short  helix that is thought to keep
the translocator closed when it is idle and to move aside when it
is engaged in passing a polypeptide chain 84

85.  In eukaryotic cells, four Sec61 complexes form a large
translocator assembly that can be visualized on ER-bound
ribosomes after detergent solubilization of the ER membrane
 It is likely that this assembly includes other membrane
complexes that associate with the translocator, such as
enzymes that modify the growing polypeptide chain, including
oligosaccharide transferase and the signal peptidase
 The assembly of a translocator with these accessory
components is called the translocon 85

86.  To release the signal sequence into the membrane, the
translocator opens laterally along the seam
 The translocator is therefore gated in two directions:
 It opens to form a pore across the membrane to let the
hydrophilic portions of proteins cross the lipid bilayer &
 It opens laterally within the membrane to let hydrophobic
portions of proteins partition into the lipid bilayer
 Lateral gating of the pore is an essential step during the
integration of transmembrane proteins
86

87. 87

88. Three ways in which protein translocation can be driven through
structurally similar translocators
88
A B C

89. The signal hypothesis
 An ER signal sequence is recognized twice
 First by an SRP in the cytosol
 Then by a binding site in the pore of the protein translocator,
where it serves as a start-transfer signal (or start-transfer
peptide) that opens the pore (e.g. for soluble proteins)
 Dual recognition may help ensure that only appropriate
proteins enter the lumen of the ER
89

90.  What happens if the pore is left open?
 The pore is a dynamic gated channel that opens only transiently
when a polypeptide chain traverses the membrane
 In an idle translocator, it is important to keep the channel
closed, so that the membrane remains impermeable to ions,
such as Ca2+, which otherwise would leak out of the ER
 As a PPC is translocating, a ring of hydrophobic amino acid side
chains is thought to provide a flexible seal to prevent ion leaks
90

91. ER translocation of proteins
 Soluble proteins
 Signal sequence has two functions
 Direct the protein to the ER
 Serves as a start-transfer signal that opens the pore
 Signal cleaved after translocation
 Pore closes & translocator opens laterally allowing the
cleaved peptide to diffuse where it is degraded
 Transmembrane proteins
 Different signals serve as start & stop transfer signal
91

92. Single-pass transmembrane protein
92
How a single-pass transmembrane protein with a
cleaved ER signal sequence is integrated into the ER
membrane

93. 93

94. Multi-pass transmembrane protein
94

95. 95
The insertion mechanism for tail-anchored proteins

96. ER translocation of proteins
 Are all proteins in the lumen resident proteins?
 Some are transit en route to other destinations
 Others are resident proteins
 Resident proteins have a retention signal of four AAs at the
C-terminal
 Catalyze protein folding: protein disulfide isomerase (PDI),
BiP (Hsp70 chaperone)
96

97.  Glycosylation of proteins in the ER
 Most important biosynthetic function of the ER
 N-linked or asparagine-linked glycosylation (Asn)
 Involves covalent addition of preformed oligosaccharides (N-
acetyl glucosamine, mannose & glucose & containing a total of
14 sugars) by oligosaccharyl transferase (membrane bound)
 Its active site exposed on the lumenal side of the ER
membrane; this explains why cytosolic proteins are not
glycosylated in this way 97

98.  A special lipid molecule called dolichol anchors the precursor
oligosaccharide in the ER membrane
 Many proteins in the cytosol & nucleus are also glycosylated, but
not with oligosaccharides:
 They carry a much simpler sugar modification, in which a single N-
acetylglucosamine group is added to a Ser/Thr of the protein
 The precursor oligosaccharide is linked to the dolichol lipid by a
high-energy pyrophosphate bond, which provides the activation
energy that drives the glycosylation reaction
98

99.  The precursor oligosaccharide is built up sugar by sugar on the
membrane-bound dolichol lipid and is then transferred to a protein
 The sugars are first activated in the cytosol by the formation of
nucleotide (UDP or GDP)-sugar intermediates, which then donate
their sugar (directly or indirectly) to the lipid in an orderly sequence
 The lipid-linked oligosaccharide is flipped, with the help of a
transporter, from the cytosolic to the luminal side of the ER
membrane
 Trimming also done in the ER (removal of 3 glucose & 1 mannose)
– marks appropriate folding and exit
99

100. Protein glycosylation in the ER
100

101. 101

102. 102

103. The role of N-linked glycosylation in ER protein folding
103

104. The export & degradation of misfolded ER proteins
104

105. 105
The unfolded protein response (UPR)

106. 106
UPR

107. Attachment of a GPI anchor to a protein in the ER
107

108. 108

109. 109

110. Intracellular Membrane Traffic
110

111. 3. Vesicular transport
 Topologically equivalent compartments of the biosynthetic-
secretory-endocytic pathways communicate through
transport vesicles
 Transport vesicles: membrane enclosed transport packages
 Vesicles continually bud off from donor compartment carrying
cargo (membrane components & soluble molecules) and fuse
with target compartment
 Each transport vesicle must be selective for the cargo to be
transported & must fuse only with the appropriate target
membrane
111

112. 112

113. 113

114. 114
Vesicle transport

115. Coat proteins
 Most vesicles formed from specialized, coated region of
membranes and bud off as coated vesicles
 Coat is discarded to allow fusion
 Coat serves two functions:
 Inner coat layer concentrates specific membrane proteins in a
specializedmembrane patches giving rise to vesicular
membrane (inner layer selects the appropriate membrane
molecules for transport)
 Outer coat layer assembles into curved like basket lattice
deforms the membrane patch and shapes the vesicle
115

116. 116

117. Coat proteins
 There are three types of coated vesicles, based on the major
coat proteins
 COPI-coated: transport from the cis Golgi cisternae to ER
(retrograde transport; transport from PM to the cell center)
and Golgi cisternae  Golgi cisternae
 COPII-coated: transport from the ER to cis Golgi cisternae
(anterograde transport; from cell center to PM)
 Clathrin-coated: transport from the trans-Golgi cisternae to
PM & from the PM to Golgi
117

118. 118
Use of different coats for different steps in vesicle traffic

119.  Protein components of clathrin-coated vesicles:
 Clathrin (major): forms the outer layer of the coat
 Each clathrin subunit consists of 3 large & 3 small polypeptide
chains that together form a three-legged structure called a
triskelion
 Clathrin triskelions assemble into a basketlike framework of
hexagons and pentagons to form coated pits (buds) on the
cytosolic surface of membranes
 Determine the geometry of the clathrin cage
 Clathrin adaptor proteins (adaptins): select cargo into clathrin-
coated vesicles 119

120. 120

121. Clathrin adaptor proteins (Adaptins – subunits)
 Vesicular transport adaptor proteins associated with clathrin
 Synthesized in the ribosomes, processed in the ER &
transported from the Golgi apparatus to the TGN & from
there via small carrier vesicles to their final destination
compartment
 The association between adaptins & clathrin are important
for vesicular cargo selection & transporting
121

122.  Clathrin coats contain both clathrin (acts as a scaffold) &
adaptor complexes that link clathrin to receptors in coated
vesicles
 Clathrin-associated protein complexes are believed to interact
with the cytoplasmic tails of membrane proteins, leading to
their selection and concentration
 Adaptor proteins are responsible for the recruitment of cargo
molecules into a growing clathrin-coated pits
122

123. Adaptins
 Heterotetrameric: composed of two large (/// & 1-4;
90-130kDa), one medium (1-4; ~50kDa), & one small (1-
4; ~20kDa) subunits
 Different pathways:
 AP-1 (/1/1/1): TGN to Endosome
 AP-2 (/2/2/2): PM to Endosome (endocytosis)
 AP-3 (/3/3/3): TGN to Lysosome
 AP-4 (/4/4/4): TGN & endosomes?
 AP-5 (?): Late Endosome to Golgi retrieval?
123

124.  Large subunits /// mediate membrane recruitment
 -adaptin of the AP-2 complex has a strong binding
preference to phosphatidylinositol 4,5-biphosphate-enriched
membranes
 -adaptin has a clathrin-binding motif & recognizes dileucine-
based sorting sequences
 -adaptin recognizes tyrosine or dileucine based sorting
sequences
 : function still unknown
124

125.  AP-2, when it binds to a specific phosphorylated
phosphatidylinositol lipid (a phosphoinositide), it alters its
conformation, exposing binding sites for cargo receptors in
the membrane
 The simultaneous binding to the cargo receptors and lipid
head groups greatly enhances the binding of AP-2 to the
membrane
 Local production of PIPs plays a major part in regulating the
assembly of clathrin coats on the plasma membrane & Golgi
125

126.  Monomeric GGA [Golgi-localizing, Gamma-adaptin ear homology,
ADP ribosylation factor (Arf)-binding proteins] adaptors
 Monomeric clathrin adaptor proteins
 Three GGAs (GGA1-3) that work at the trans-Golgi network, in
endosomes to sort transmembrane cargo proteins such as
mannose 6-phosphate receptors, sortilin, β-site amyloid precursor
protein cleaving enzyme 1, and epidermal growth factor receptor
 The cytoplasmic regions of these cargoes possess motifs of acidic
amino acid cluster-dileucine and/or ubiquitination sites, which can
be recognized by GGAs
126

127. Adaptins
Lipid-induced conformation switching of AP2
127

128. Control of Coat Assembly
 Monomeric GTPases (coat-recruitment GTPases) control coat
assembly
 Coat proteins must assemble only when and where they are
needed
 To balance the vesicle traffic to and from a compartment
 Coat-recruitment GTPases, control the assembly of clathrin
coats on endosomes & COPI & COPII coats on Golgi & ER
membranes
128

129.  ARF proteins: responsible for the assembly of both COPI &
clathrin coats at Golgi membranes
 Sar1 protein: responsible for the assembly of COPII coats at
the ER membrane
 Coat-recruitment GTPases are usually found in high
concentration in the cytosol in an inactive, GDP-bound state
129

130.  When a COPII-coated vesicle is to bud from the ER
membrane, for example, a specific Sar1-GEF embedded in
the ER membrane binds to cytosolic Sar1, causing the Sar1
to release its GDP & bind GTP in its place
 In its GTP-bound state, the Sar1 protein exposes an
amphiphilic helix, which inserts into the cytoplasmic leaflet of
the lipid bilayer of the ER membrane
 The tightly bound Sar1 now recruits adaptor coat protein
subunits to the ER membrane to initiate budding
130

131.  The coat-recruitment GTPases also have a role in coat disassembly
 The hydrolysis of bound GTP to GDP causes the GTPase to change
its conformation so that its hydrophobic tail pops out of the
membrane, causing the vesicle’s coat to disassemble
 When the vesicle docks at a target membrane, a kinase
phosphorylates the coat proteins, which completes coat
disassembly and readies the vesicle for fusion
 Clathrin- and COPI-coated vesicles, by contrast, shed their coat
soon after they pinch off
 For COPI vesicles, the curvature of the vesicle membrane serves
as a trigger to begin uncoating
131

132. 132

133.  Budding of a transport vesicle
 Polymerization of a protein coat induces formation of a
vesicle
 Process initiated and probably regulated by a specific GTP-
binding protein in collaboration with phosphoinositides
 Interactions between coat proteins and the cytosolic domain
of membrane proteins allow selective incorporation of
proteins into the vesicle
 Vesicle contains proteins that target it to its correct
destination
 Protein coat depolymerized after vesicle is formed 133

134. Vesicle formation
134

135. Clathrin assembly
• Dynamin assemble as a ring around the neck of each bud
- PI (4,5)P2-binding domain tethers to membrane
- GTPase regulates rate of vesicle pinching
- Bend the patch of membrane
 Distorting the bilayer structure or changing its lipid
composition
 Phosphatase (synaptojanin), HSP70, auxillin & endophilin
involved in vesicle uncoating 135

136. 136
Formation of clathrin vesicles
Dynamin

137. 137

138. COPII assembly
 Coat-recruitment GTPases (monomeric) control the assembly
of clathrin, COPI & COPII coats
 GEF binds to a GTPase
 GTPase exposes its hydrophobic tail
 Also activate phospholipase D to generate phosphatidic acid
 GTPase recruit coat proteins to initiate budding
 Protein-protein & protein-lipid interaction deforms the
membrane
 Pinching-off the vesicle
138

139. 139

140. 140

141. 141
Formation of a COPII coated vesicle

142. 142
Not all transport vesicles are spherical
 Packaging of procollagen into large tubular COPII-coated vesicles

143. How specificity in targeting is assured?
 Transport vesicles display surface markers (origin and type of
cargo)
 Target membranes display complimentary receptors
 Process controlled by two classes of proteins
- SNAREs: vSNAREs & tSNAREs
 Provide specificity and catalyze fusion
- Targeting GTPases: Rab
 Regulate initial docking and tethering
143

144.  Rab proteins (Ras-associated binding proteins):
 Largest subfamily of monomeric G-proteins (over 60 known
family members)
 They cycle between cytosol & d/t membranes & in GTP/GDP
bound form
 Regulate the reversible assembly of protein complexes at the
membrane
 Guide transport vesicles to their target membrane
144

145. 145
Subcellular Locations of Some Rab Proteins

146.  Determine rate of vesicle docking and matching of SNAREs
 Bind to different proteins
 Rab-GDP dissociation inhibitor or GDI: keep Rab proteins
inactive
 Rab effectors/Rab-GEFs: activate Rab proteins
 Motor proteins: propel vesicles along actin filaments
 Tethering proteins: link two membranes
o Interact with SNAREs to couple membrane tethering to fusion
146

147. Vesicular transport
The formation of a Rab5 domain on the endosome membrane
147

148. A model for a generic Rab cascade
The Rab cascade: changes organelle identity 148

149. Tethering of a transport vesicle to a target membrane
149

150. 150

151. 151

152.  NSF: N-ethylmaliemide sensitive fusion protein (Sec18)
 SNAP: Soluble NSF attachment protein 152

153. 153

154. Postulated role of Rab proteins in facilitating the docking of
transport vesicles
154

155. Vesicular transport
 Docking vs fusion:
 Vesicle once docked unloads its cargo by membrane fusion
 Docking may not be however always be followed by
immediate fusion
 In regulated exocytosis, fusion is delayed until triggered by
specific extracellular signal
 Docking: proteins adhere and interact
 Fusion: lipids flow from one bilayer to the other
155

156.  Fusion requires the displacement of water from the
hydrophilic surface of the membrane
 When SNAREs form a coiled coil the energy released helps to
squeeze water molecule from the interface
156

157. SNARE proteins may catalyze membrane fusion
157

158. 158

159. A) Transport from ER to Golgi
 Biosynthetic-secretory pathway is initiated from ER
 Only folded proteins are allowed to exit & folding is assisted
by ER chaperones
 Cargo carried by COPII vesicles and bud from region called ER
exit sites
 Budded vesicles fuse and form vesicular-tubular cluster (VTC),
also referred to as the ER-Golgi intermediate compartment
(ERGIC)
 Homotypic fusion or heterotypic fusion
159

160. 160

161. 161
Vesicular tubular clusters

162. ER to Golgi
 ERGIC bud vesicles (COPI) that retrogradely transport ER
resident proteins having ER retrieval signal
 Found at the C-terminal
 KKXX: displayed by ER membrane proteins and bind directly
to COPI coats and packaged
 KDEL: soluble proteins (e.g. BiP) and bind to specialized
receptors (KDEL receptor)
162

163. Retrieval of soluble ER resident proteins
163

164. ER to Golgi
 KDEL receptor packages returning proteins that display KDEL
sequence into COPI vesicles
 Receptor shuttles between ER & GA and has different affinity
for KDEL sequences at the two compartments
 Bind KDEL sequences at the slightly acidic pH in ERGIC & GA
and releases at the neutral pH at the ER
 Kin recognition (aggregation of proteins that function in the
same compartment) is another mechanism used to retain ER
proteins (appears to be major mechanism)
164

165. KKXX sequence
at the C-terminus
attaches to COPI
165

166. Transport through the Golgi
 GA physically and functionally linked to the ER
 Consists of a collection of flattened, membrane enclosed
cisternae somewhat resembling a stack of pancakes
 A series of such cisternae is called a Golgi stack (dictyosome
in plants)
 4-6 cisternae per stack, number and size of the stack vary
with cell type and metabolic activity
 Golgi is surrounded by numerous transport vesicles
166

167. Transport through the Golgi
 Each Golgi stack has two faces
 The cis (entry or forming): oriented towards the transitional ER
 The trans (maturing or exit)
 Both faces are associated with a special compartment called
CGN and TGN, respectively
 The sac between the CGN & TGN is called the medial
cisternae (much of protein processing occur here)
167

168. 168

169. Through the Golgi
 CGN, TGN & medial cisternae are functionally & biochemically
distinct
 Enzymes and receptors they harbor for protein processing
 Kind of coated vesicles
 From ER are COPII
 From CGN or medial cisternae are COPI
 From TGN are clathrin
169

170. 170

171. Through the Golgi
 O-glycosylation occurs in the GA
 Oligosaccharide processing steps occur in an organized
sequence
 Each cisterna has an abundance of specific enzymes
 Prominent in cells specialized in secreting glycoproteins
 Large vesicles are found in the trans side
 Two of the most important categories of enzymes are glucan
synthetases and glycosyl transferases 171

172. Through the Golgi
 Terminal to distinguish it from core glycosylation
 Occurs in the luminal not the cystosolic side
 Includes removal, addition or no further processing
 Two major classes of N-linked oligosaccharides
 Complex oligosaccharides: trimming & further addition of
GluNAc & other CHO (gal, sialic acid, fructose)
 High mannose oligosaccharides: trimming but no addition,
retain 2 GluNAc & 6-8 mannose 172

173. 173
Oligosaccharide processing in the ER & Golgi

174. Through the Golgi
 Proximity to enzymes determines type
 O-glycosylation: mucin and proteoglycan core protein
 Purpose of glycosylation
 Promotes protein folding ( solubility and binding)
 Marker for complete folding in the ER
 Imparts resistance to digestion
 Cell adhesion
 Signaling, recognition by cargo receptors (ERGIC53 lectin)
174

175. ER to Golgi
 How do Golgi resident proteins are recognized?
 May have retention or retrieval tags & Kin recognition
 GC proteins are membrane bound proteins
 The length of membrane spanning domains may determine
to which cisterna of the GC the protein settles
  in length of membrane spanning domain from CGN to the
TGN
175

176. ER to Golgi
 Mechanism of movement of molecules from one cisternae to
the other is unknown
- Two major hypotheses
 Vesicular transport model
 Considers Golgi as static structure
 Cisternal maturation model
 Considers Golgi as dynamic structure
176

177. 177

178. 178

179. 179

180. 180

181. B) Transport from TGN to lysosomes
 Structures filled with hydrolytic enzymes
 About 40 types of acid hydrolases
 Need proteolytic activation and acid environment
 A vacuolar H+ ATPase maintains luminal pH acidic
 Lysosomal membranes are highly glycosylated
 Lysosomes are diversely heterogeneous
 Wide variety of digestive function
 The way lysosomes form 181

182. 182
Lysosomes

183. Lysosome maturation model
183

184. TGN to lysosomes
 Three pathways deliver materials to lysosomes
- Endocytosis: EE-LE-LYSO, clathrin mediated
- Authophagy: endosome-mediated (?)
 Engulfing by nucleation & extension of a delimiting
membrane
 Closure of the autophagosome into a sealed double
membrane bounded compartment
184

185. TGN to lysosomes
 Fusion with lysosome
 Digestion of the membrane and its contents
- Phagocytosis (actin-mediated)
 Phagosome fuses with lysosome
185

186. 186
Four pathways to degradation in lysosomes

187. A Model of Autophagy
187

188. TGN to lysosomes
 Cargoes destined are sorted in the TGN and are recognized
by the marker mannose-6-phosphate
 Two Golgi-specific enzymes catalyze the process
 Phosphotransferase (cis golgi): adds GlcNAc-phosphate (I-
cell disease; a lysosomal storage disease)
 The second (trans golgi): removes GlcNAc
 Mannose-6-phosphate receptor binds at pH 6.5-6.7 (TGN)
and releases at pH 6 (endosome)
188

189. The Recognition of a lysosomal hydrolase
189

190. Transport of newly synthesized lysosomal hydrolases to endosomes
190

191. Endocytosis
 A process by which cells take up macromolecules & even
other cells by means of membrane budding
 May encompass several processes differing in nature of
material ingested or mechanism employed
 Separate the material ingested from the cytosol
 Most fuse with an early endosome, adding their contents to
the stream of material moving to lysosome
191

192.  Alternatively, may form temporary connection with
endosomes, acquiring digestive enzymes & maturing to
lysosomes
 Two main types
 Phagocytosis (cellular eating, phagosomes)
 Pinocytosis (cellular drinking)
192
Endocytosis

193. Endosome maturation: the endocytic pathway from PM to lysosomes
193

194.  Important for nutrition in protozoa, worms
 Other purposes (defense) in animal cells and carried out by
specialized cells (phagocytes)
 Two major types of phagocytes
 Macrophages: scavenge senescent and dead cells, ingesting
cellular debris and whole damaged cells
 Neutrophils
194
Phagocytosis

195. Phagocytosis
 Other cells engaged in phagocytosis:
 Fibroblasts: take up collagen to allow remodeling of the tissue
 Dendritic cells: ingest bacteria in spleen
 A triggered process:
 Antibodies
 Sticking out of phosphatidyl serine to the EC side of
membranes; apoptosis
195

196. Phagocytosis
 Process involves
 Formation of pseudopods
 Pseudopods engulf & forming an intracellular phagocytic
vacuole (phagosome)
 Fuse with late endosome or mature to lysosome
 Phagocytes generate hydrogen peroxide, hypochlorus acid
and other oxidants in the phagosome to kill micro-organisms
196

197. Phagocytosis
 Phagocytes do not eat live cells
 Display a do not eat me signal
 Surface proteins interact with inhibitory receptors on
phagocytes
 Activating a phosphatase
 Antagonize the signaling event required for initiation of
phagocytosis
197

198. Pinocytosis
 Two types:
i) Clathrin dependent (receptor-mediated endocytosis)
 Process by which an external ligand binds to a receptor on
the surface of the cell is internalized
 Provides a selective concentration mechanism that increases
the efficiency of internalization
 Phagocytosis does not concentrate & not clathrin-dependent
198

199. Pinocytosis
 Begins at clathrin coated pits
 Invagination and pinching off
 First described by uptake of cholesterol
 All animal cells require cholesterol for their membrane
 But cannot tolerate excess in the blood
 Therefore is transported as a complex
 The most abundant being LDL
199

200. 200

201. 201
The receptor-mediated endocytosis of LDL

202. Pinocytosis
 Fate of endocytosed receptor
 Recycled & return to the same domain of the PM (LDL
receptor)
 Proceed to a different domain of the PM thereby mediating a
process called transcytosis
 Progress to lysosome where they are degraded (receptor
down regulation)
 Fate of ligand
 Ligand degraded (e.g. LDL) or recycled (tranferrin receptor &
apotransferrin)
202

203. Possible fates for transmembrane receptor proteins that
have been endocytosed
203
Fc: fragment crystallizable

204. Pinocytosis
ii) Clathrin-independent endocytosis
 Fluid-phase endocytosis
 Non-specific internalization of extracellular fluid (ECF)
 Substances dissolved in the ECF can also be internalized
 Does not concentrate ingested material
 Means for controlling cell volume and surface area
204

205. 205
Macropinocytosis.

206. Pinocytosis
 Caveolar pathway
 Transport molecules across the endothelial cells
 Formed from membrane micro domains (lipid rafts)
 Major protein: Caveolins (integral proteins)
 Form vesicles by virtue of lipid composition
 Vesicles pinch off using dynamin
 Deliver their contents to caveosome or transcytosis
206

207. Pinocytosis
Uptake of cholera toxin and SV40
207

208. Transcytosis
 Receptors on the polarized epithelial cells transfer
macromolecules from one EC space to another by this process
 Best e.g. is passive transfer of antibody (IgG) to the newborn
 Cells can also regulate the flux of proteins by transcytotic
pathway according to need
 E.g. Storage of GLUT in recycling endosomes by muscle and fat
cells until stimulated by insulin
208

209. 209

210. Storage of PM protein in recycling endosome
210

211. Exocytosis
 Fusion of transport vesicles containing molecules with the PM
to be secreted to the cell exterior
 Two pathways identified:
 Constitutive secretory pathway (default pathway)
 Common to all cells (e.g. secretion of mucus)
 Regulated secretory pathway
 Common in cells specialized for secreting products rapidly on
demand (glands, neurons)
211

212. Exocytosis
212

213. Budding of secretory vesicles (on demand)
 Secreted molecule stored in secretory vesicles (secretory
granules or dense core vesicles)
 Secretory proteins are packed in the TGN by selective
aggregation (signal patches responsible)
 But not known how they are disaggregated into secretory
vesicles (receptor in vesicles recognize signal patches??)
 pH in the TGN and secretory vesicles favors aggregation
213

214.  Leave the TGN as immature secretory vesicles and mature in
due course
 Retrieval of membrane, progressive acidification,
condensation, and proteolytic processing
 Contain much more highly concentrated protein than
constitutive
 Form large protein aggregates that exclude non-secretory
proteins 214
On demand...

215. On demand...
 The molecule released in response to EC signals
 Signal is often a hormone/neurotransmitter /IC Ca2+
 In neurons, synaptotagmin, a Ca2+ sensor enhances binding
of vSNAREs and tSNAREs
 Synaptotagmin reduces activation energy by inducing high
positive curvature in the membrane
 Vesicle fusion es surface area but removed by endocytosis
215

216. Polarized secretion
 PM may have different domains
 Delivery from TGN should maintain this difference
 Strategy for secretion
 Random delivery followed by selective retention or removal
 TGN separate and dispatch in vesicles
216

217. Constitutive & regulated secretory pathways
217

218. 218
The 3 best-understood pathways of protein sorting in the TGN

219. Traffic through the endomembrane system
219