1Outline:
pathway
3. Vesicular trafficking in the early stages of
the secretory pathways
of the secretory pathways
sorting of internalized proteins
6. Synaptic vesicle function and formation
SEM of the formation of clathrin-coated vesicles on the cytosolic face of the plasma membrane
Secretory pathway: protein to various organelles by transport vesicles Anterograde: forward moving Retrograde: backward moving Trans position: farthest from the ER Cis position: nearest the ER Cisternal progression: cis-Golgi cisterna → cargo of protein → move form cis → medial → trans ; anterograde transport vesicle; normal TGN (trans Golgi network): proteins not transport to ER or Golgi, are destined for compartment to others (by different types of vesicles) 1. from trans → fuses membrane → trnasport → exocytosis 2. from trans → stored inside → formation of secretory vesicles;
release by signal for exocytosis 3. from trans → late endosome → lysosome (intracellular
degradation of organelle) the mechanism not well know endosome had endocytic pathway, from the plasma membrane
bringing membrane proteins and their bound ligands into the cell
transport vesicle cargo proteins same orientation anterograde transport vesicles retrograde transport vesicles cisternal progression trans-Golgi network (TGN) secretory vesicle (regulated..) constitutive secretion-exocytosis transport vesicle-late endosome endocytosis
Overview of secretory & endocytic pathways: Transport vesicles
Pulse-chase labeling & EM autoradiography
Tissue sections of pancreas acinar cells -> a brief incubation (3 min) with H3-Leucine -> transfer to unlabeled medium & incubate for a period of time (0, 7, 37, 117 min) -> cover tissue sections with photographic emulsion - > EM
17.1 Techniques for studying the secretory pathway:
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17.1 Techniques for studying the secretory pathway:
At restrictive temp. of 40oC, newly made G protein is misfolded & retained within ER.
At permissive temp. of 32oC, accumulated G protein is correctly folded & transported through secretory pathway.
Different time course → change Temp → misfolded → stop transport
Palade’s early exp had found that in mammalian, vesicle mediated transport of a protein molecule from ER to membrane about 30-60 min.
17.1 Techniques for studying the secretory pathway: by living cells
1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide
modifications 2. Yeast mutants define major stages and many components in
vesicular transport 3. Cell-free transport assays allow dissection of individual steps in
vesicular transport
Fig17-2 Protein transport through the secretory pathway can be visualized by fluorescence microscopy of cells producing a GFP-tagged membrane
protein: VSV G protein
Use temperature-sensitive mutant, VSVG-GFP. 40oC the protein in ER 32oC move → Golgi → plasma membrane→
Form ER to Golgi about 60min
17.1 Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide
modifications 2. Yeast mutants define major stages and many components in
vesicular transport 3. Cell-free transport assays allow dissection of individual steps in
vesicular transport
Addition & processing of N-linked oligosaccharides in R-ER of vertebrate cells
• glycosidases (cis-) • endoglycosidase D
Cleavage by endoglycosidase D. In cis, specific glycosidaseRemove 3 mannose
Remove 2 mannoseAdd
Cleavage by endoglycosidase D
Cell expression VSV G protein → at Temp 40 → link radioactive aa and protein keep in ER → Tem 32 C → VSV G extracted → digested by endoglycosidase (about cis Golgi protein) → SDS electrophoresis
Endoglycosidase can not cleavage ER’s protein.
32 C: protein move from ER → Golgi (modification) → membrane
40 C: in ER not move.
Protein folding ok → move → golgi → can cleavage
From ER to golgi about 60 mi
17.1 Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide
modifications 2. Yeast mutants define major stages and many components in
vesicular transport 3. Cell-free transport assays allow dissection of individual steps
in vesicular transport
4
Fig 17-5 Phenotypes of yeast sec mutants identified stages in the secretory pathway
Yeast sec (secretion) mutants
The temperature sensitive mutant → grouped into 5 classes Combination of different mutant → for research of protein transport pathway, ie BD → protein in ER not Golgi → so ER is before, and Golgi is after. These studies confirmed that: cytosol → RER → ER-to Golgi transport vesiceles → Golgi cisternce → secretory → exocytosed
protein 17.1 Techniques for studying the secretory pathway:
1. Transport of a protein through the secretory pathway can be assayed in living cells: 1) Microscopy of GFP-labeled VSV G protein 2) Detection of compartment-specific oligosaccharide
modifications 2. Yeast mutants define major stages and many
components in vesicular transport 3. Cell-free transport assays allow dissection of individual
steps in vesicular transport
Fig 17-6 Protein transport from Golgi cisternae to another can be assayed in a cell- free system
Cell-free transport assay
Can not add
Proof: golgi can retrograde vesicular transport for midification
5
Trans-Golgi Progression
Tradional Model - Golgi is a static organelle. Secretory proteins move forward in small vesicles. Golgi resident proteins stay where they are.
“Radical” Model - Golgi is a dynamic structure. It only exists as a steady-state representation of transport intermediates. Secreted molecules move ahead with a cisterna. Golgi resident proteins move backward to stay in the same relative position.
17.2 Molecular mechanisms of vesicular traffic
Fig 17-7 Overview of vesicle budding and fusion with target membrane
(a) Coated vesicle: From membrane interaction with integral (b) Uncoated vesicle: Target membrane
vSNARE: Crucial to fusion of the vesicle with correct target membrane tSNARE: specific joining of vSNARE
Vesicle transport: from organelle (Donor) target organelle
Assembly of a protein coat drives vesicle formation & selection of cargo molecules.
A conserved set of GTPase switch proteins controls assembly of different vesicle coats
Three types of coated vesicles have been characterized. All need GTP binding
GTPase superfamily
from the plasma membrane and trans-Golgi network to late
endosomes
– With AP2: Endocytosis (PM to endosome)
– With AP3: Golgi to lysosome and other vesicles
COPI: Golgi to ER (retrograde transport)
COPII: ER to Golgi (antrograde trnasport)
AP: complex consists of four different subunits
Vesicle buds can be visualized during in vitro budding reactions.
Coated vesicles Artifical membranes and purified coat protein (COP II) → polymerization of coat protein onto the cytosolic face of the parent membrnae
A conserved set of GTPase switch proteins controls assembly of different vesicle coats.
All three coated vesicles contain a small GTP-binding protein
COP I and clathrin vesicle: ARF (ADP-ribosylation factors)
COP II vesicle: Sar I protein
ARF and Sar I protein can switch GTP (GDP-protein → GTP-protein
active)
There two sets of small GTP-binding proteins for vesicle secretion. One
is ARF and Sar I; another is Rab protein
ARF (ADP Ribosylation Factor) protein exchanges bound GDP for GTP and then binds to its receptor on Golgi membrane
A conserved set of GTPase switch proteins controls assembly of different vesicle coats.COPII coated formation
GTP → Sar1 conformational change →Sar1-GTP binding to membrane → polymerization of cytosolic complexes of COPII subunit on the membrane → formation of vesicle buds
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Monomeric GTPase control coat assembly
Sar1 attached to Sec23/24 coat protein complex → cargo protein are recruited to the formation vesicle bud by binding of specific short sequence in their cytosolic regions to sites on the Sec23/24 → assembly to second type of coat complex composed of Sec13/31 → completed → Sec23 promotes Sar1-GTP hydrolysis → release Sar1-GDP → disassembly of the coat → transport vesicle
Cargo protein
Specific receptor
Major coat protein: clathrin & adaptin There are at least four types of adaptins, each specific for a different set of cargo receptor.
by charperone (hsp70)
Vesicle formation
Coat assembly controlled by monomeric G-protein (SAR1 or ARF) with fatty acid tail
GDP-bound SAR1 or ARF are free in cytosol
Membrane-bound G-protein recruits coat protein subunits
Assembly of coat pulls membrane into bud Leads to exposure of fatty acid tail membrane binding Donor membrane contains guanine nucleotide-releasing factor -causes Sar1-GDP SAR1-GTP
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Coated vesicles accumulate during in vitro budding reactions in the presence of a nonhydrolyzable analog of GTP
Golgi membrane + COPI coat proteins and GTP → bud off Non-hydrolyzable GTP prevent disassembly of the coat after vesicle release
Targeting sequence on cargo proteins make specific molecular contacts with coat protein
The retrieval pathway to the ER uses sorting signals
Lys-Asp-Glu-Leu (KDEL)
Short retrieval signal at c-terminal
Resident ER membrane protein
Different Rab GTPases & Rab effectors control docking of different vesicles on target membranes: vesicle docking
controlled by Rab protein.
Monomeric GTPases attach to surface of budding vesicle
Rab-GTP on vesicle interacts with Rab effector on target membrane
After vesicle fusion GTP hydrolysed, triggering release of Rab-GDP
Different Rab proteins found associated with different membrane-bound organelles
v-SNARE
t-SNARE
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Monomeric Rab-GTPases A guanine nucleotide exchange factor (GEF) recognizes a specific rab proteins and promotes exchange of GDP for GTP.
GTP bound Rabs have a different conformation that is the “active” state.
Activated rabs release GDI, attach to the membrane via covalently attached lipid groups at their C-termini and are incorporated into transport vesicles.
Rab-GTP recruits effectors that can promote vesicle formation, vesicle transport on microtubules, and vesicle fusion with target membranes.
After fusion Rab-GTP hydrolyzes GTP to GDP and is released from the membrane. GTPase activating proteins proteins accelerate hydrolysis, reducing the avalability of active rabs.
Rab proteins (monomeric GTPase) help ensure the specificity of vesicle docking
Paired sets of SNARE proteins mediates fusion of vesicles with target membranes.
Analysis of yeast sec mutants defective in each of the >20 SNARE genes.
In vitro liposome fusion assay. SNARE-mediated fusion →
exocytosis → secretory protein In this case, v-SNARE as VAMP
(vesicle associated membrane protein)
hydorphobic anchor. Formation of four-helix bundle:
VAMP (1), Syntaxin (1) and SNAP- 25 (2)
But, in COPII with cis, each SNARE has provide one helix SNARE complex had specificity
Dissociation of SNARE complexes after membrane fusion is driven by ATP hydrolysis.
SNARE complex formation by non-covalent interaction. Dissociate → free SNARE → can fuse next time
Two protein play important role of dissociation or fusion with a target membrane: NSF (NEM- sensitive factor, blocked by N-ethylmaleimide) & α- SNAP (soluble NSF attachment protein). hexamer
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NSF or n-ethylmaleimide (NEM) Sensitive Factor
SNAP- Soluble NSF Attachment Proteins NSF + SNAP bind to target membranes (synaptic vesicle & plasma membrane)
Receptors for NSF and SNAP are synaptobrevin (vesicle), SNAP- 25 (plasma membrane) and syntaxin (plasma membrane)
Membrane targets are called SNAREs (v- and t-) Soluble NSF Attachment protein REceptors
SNAP-25- Synaptosome Associated Protein of 25 kDa • Over-expression of truncated SNAP-25 blocks release • Syntaxin, 15 kDa protein • Sensitive to botulinum toxin A cleavage - release prevented
Identified and cloned ~ 1988-1990
and sometimes abbreviated as Syb
Cleaved by tetanus toxin (failure of exocytosis = death)
Spans vesicle membrane
Synaptobrevin
Dissociation of SNARE complexes after membrane fusion is driven by ATP hydrolysis.
ATP is not actually required for release once vesicles are docked, but is thought to break down the SNARE complexes to promote recycling.
Rizo and Sudhof 2002 Nature Rev. Neurosci.
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Rizo and Sudhof 2002 Nature Rev. Neurosci.
Membrane fusion reactions need to overcome repulsive forces that take over when membranes approach within 3nm- hydration for ectoplasmic and cytoplasmic leaflets as well as charge repulsion in cytoplasmic leaflets. Attractive hydrophobic forces can be enhanced by membrane bending.
Rab proteins (monomeric GTPase) help ensure the specificity of vesicle docking
Specificity of vesicle fusion
Need mechanism for selective vesicle trafficking -controlled by SNAREs and Rab proteins
SNARE hypothesis proposes specific interactions between v- SNAREs and t- SNAREs govern vesicle docking and fusion
Each organelle has specific SNAREs leading to specific vesicle fusion
Vesicle docking controlled by Rab proteins
Monomeric GTPases attach to surface of budding vesicle
Rab-GTP on vesicle interacts with Rabeffector on target membrane
After vesicle fusion GTP hydrolysed, triggering release of Rab-GDP
Different Rab proteins found associated with different membrane-bound organelles
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Summary
folded, enclosed in vesicles -proteins only have to cross ER
membrane
and plasma membrane
Cargo selected by sorting/cargo receptors
Specificity of fusion controlled by Rabproteins, v-SNAREs and t-
SNAREs
The structure of influenza hemagglutinin (HA)
Three HA1 and three HA2
HA1
Conformational changes in influenza HA protein trigger membrane fusion
Virus binds to cell surface receptors modified with sialic acid. The “fusion peptide” is buried within the HA protein at neutral pH. (Spring-Loaded) The virus enters the endosomal pathway where the pH is lower. At pH 5 HA protein undergoes radical conformational change, extending the hyrophobic “fusion-peptide” into the target membrane, initiating fusion, releasing the viral DNA into the cytoplasm. The V-SNARE/T-SNARE/SNAP25 “snare-pin” resembles the HA “hairpin” .
Binding of fusion peptide to HA2 disrupted. Globular domains dissociate.
Loop segment forms a continuous helix.
Fusion peptide inserts into endosomal membrane.
Membrane fusion machines: membrane fusion is catalyzed by intrinsic membrane proteins that undergo assembly in trans across fusion partner membranes. Influenza hemaglutinin protein allows fusion of viral membrane with endosome upon pH-induced conformational change.
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Viral fusion proteins and SNAREs may use similar strategies Model for membrane fusion directed by hemagglutinin (HA)
Need mechanism for selective vesicle trafficking -controlled by SNAREs and Rab proteins
SNARE hypothesis proposes specific interactions between v- SNAREs and t-SNAREs govern vesicle docking and fusion Each organelle has specific SNAREs leading to specific vesicle fusion
17.3 Early stages of the secretory pathway
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Fig 17-14 Vesicle-mediated protein trafficking between the ER and cis-Golgi
Anterograde-COPII vesicle Retrograde-COPI vesicle
Cargo protein vSNAREs (yellow)
COPII vesicles mediate transport from the ER to the Golgi
3-D structure of ternary complex comprising the COPII coat proteins (Sec23, Sec24) & Sar1-GTP.
Formation of COPII vesicles: triggered by Sec12 → induced catalyzes the GDP for GTP of Sar1 → binding Sar1 to ER membrane → followed by binding of Sec13/24 → formation of complex →second complex comprising Sec13 and 31 → interact with fibrous proteins Sec 16 → coat polymerization
Sec24: interact with integral ER → transport to Golgi
Di-acidic sorting signal (Asp-X-Glu, or DXE).
ER lumen cytosol
COPI vesicles mediate retrograde transport within the Golgi and from the Golgi to the ER
Most soluble ER-resident protein carry a Lys-Asp-Glu-Leu (KDEL) sequence at C-terminus.
KDEL signal & KDEL receptor: retrieval of ER-resident luminal proteins from Golgi.
Both COPI and II vesicle had KDEL receptor.
Retrieval system prevented ER luminal protein for folding.
KDEL binding affinity is sensitive pH. It binding protein in Golgi, but release in ER.
PH high
KDEL-receptors bind to KDEL-bearing proteins in the low pH environment of the Golgi and release that Cargo in the neutral pH of the ER.
pH probably alters KDEL receptor conformation - regulating cargo binding and inclusion in COPI vesicles.
15
COP I vesicles mediate retrograde transport for retrieval of ER resident proteins (recycle protein)
necessary for soluble secretory proteins to move anterograde without loss of ER resident proteins (e.g., PDI, BiP)
ER resident proteins possess ER retrieval signals – KKXX at C-terminal end for ER membrane proteins interacts w/
COP1α/β (e.g., PDI) – KDEL at C-terminal end for ER soluble proteins interacts w/ KDEL receptor
(e.g., BiP) KDEL receptor serves to retrieve KDEL tagged proteins from cis-Golgi and
return them to ER – KDEL receptors localized primarily to membranes of cis-Golgi itself and to
small vesicles that shuttle between ER and cis-Golgi KDEL and KKXX signals are both necessary and sufficient for ER retention
Lys-Lys-X-X in KDEL receptor or membrane receptor( Retrieval of ER- resident membrane proteins from Golgi)
At the very end of C-terminus, which faces the cytosol. Binds to COPI α & β subunits and retrograde to ER.
Anterograde transport through the Golgi occurs by cisternal progression
Cisternal progression: protein form cis to trans Trans more large than cis
Anterograde transport through the Golgi occurs by cisternal progression.
Large macromolecular assemblies (e.g. algal scales & precollagen aggregates) are too large and never found in transport vesicles.
COP II
COP I
clathrin
– TGN → late endosome (e.g., lysosomal targeting)
16
17.4 Later stages of the secretory pathway
Three major types of coated vesicles in secretory & endocytic pathways. COPII: mediate anterograde
transport from ER to cis Golgi complex.
COPI: retrograde transport from cis to ER
Secretory proteins: coated protein (usually clathrin), move from cis to trans, is also cisternal progression.
trans-Golgi network (TGN)
Vesicles coated with clathrin and adapter proteins mediate several transport steps (clathrin-coated vesicle; CCV)
Structure of clathrin coats (36 triskelions)
Trans-Golgi →Vesicles, has two layered, outer composed of the fibrous protein clathrin and inner layer compose of adapter protein (AP) complex.
3 heavy (180k) and 3 light (35-40k) chain → triskelion ()
Fibrous cathrin coat around vesicles is constructed of 36 clathrin triskelion
AP complex determine which cargo protein specifically to included in or excluded from. AP: has 1, 2, 3 subunit
All vesicles, ARF → initiate coat assembly →onto the membrane
AP and GGA bind to the cytosolic domain of cargo protein.
Clathrin-coated vesicles
Clathrin/AP (adaptor protein complex) Clathrin/GGA1/2/3
– GGA identified in 2000 – 3 GGAs in human, 2 in yeast
adaptin complexes recognize specific cargo and link it to clathrin assembly
Types of AP complexes: AP1, AP2, AP3, GGA.
Tyr-X-X-φ signal sequence (φ hydrophobic), from cis-golgi budding; interac with AP1 GGA
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GGAs Golgi-associated, γ-adaptin ear homologous, ARF-binding proteins
Nakayama Nakayama et al.et al., , Cell Structure and Function 28:Cell Structure and Function 28: 431431--442. (2003) 442. (2003)
APAP--11
HingeHinge
GGAGGA
Localized to the trans-Golgi network (TGN) Transport to the endosome/lysosome system
GGAs are clathrin adaptors
GGA domains and function
GAT: ARF-interacting and Golgi localization
Hinge: clathrin-binding GAE: binding accessory
proteins
Robinson Robinson et al.et al., , Current Opinion in Cell biology 13:Current Opinion in Cell biology 13: 444444--453. (2001) 453. (2001)
mannose 6- phosphate receptor
• GGA inhibits ARF hydrolysis • GGA has a docking site via
M6PR • ARF dissociates before
disassembly
by cargo receptor • COPI associates with
membrane through ARF • COPI promotes ARF
hydrolysis • ARF hydrolysis drives coat
protein dissociation
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Dynamin is required for pinching off () of clathrin vesicles
Model for dynamin-mediated pinching off of clathrin/AP-coated vesicles
Dynamin is needed for left donor membrane, need GTP hydorlysis COPI and II did not need
Fig 17-21 GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles in cell free extract (GTP-γ-S)
No GTP hydrolysis → no pinching off of clathrin-coated vesicles
Lysosomes and cellular digestion
materials (endocytosis), intracellular materials and macromolecules
The endpoint of the endocytosis pathway for many molecules is the lysosome, a highly acidic organelle rich in degradative enzymes.
The V-ATPase maintains the high acidity of the lumen by pumping protons across the lipid bilayer.
Lysosomes isolate digestive enzymes from the rest of the cell
To be discovered in 1950s Enzymes: acid phosphatase, beta-glucuronidase,
deoxyribonuclease, ribonuclease, protease Containing various size and shape (generally ~0.5 μm in
diameter) A single membrane Have ATP-dependent proton pumps to maintain pH value
(4.0-5.0) → for denature and degradation of macromolecules → actively or passively transport → to cytosol
Major enzymes are acid hydrolases – Could digestive entire organelles – Could not digestive lysosomal membrane by glycosylation of
interior membrane
Lysosomes develop form endososomes
Lysosomal enzymes : synthesized in RER → golgi → sorted in TGN → have mannose-6-phosphate → packaged in clathrin-coated vesicles → budded from TGN → to one of the endosomal compartments (early endosome) → late endosome (full complement of acid hydrolases) →proton pump → change pH
Enzymes activation mechanisms
– Moving the enzymes
– More acidic environment
– Transfer material to an existing lysosome
Lysosomal enzymes are important for several different digestive processes
Functions of lysosomes – Nutrition – Defense – Recycling of cellular components – Differentiation – Phagocytosis, receptor-mediated endocytosis, autophagy,
extracellular digestion – Autophagy: The original recycling system
What are the functions of and pathways to the lysosome?
Vesicular transport from the cell membrane -- endocytosis, phagocytosis vs pinocytosis
Autophagy: The original recycling system
Breakdown of cellular structures and components (old, damage, no longer need)
Two types – Macrophagy: a double membrane organelle that derived
from the ER → autophagic vacuole (or autophagosome) – Microphagy: a single phospholipid bilayer that encloses
small bits of cytoplasm rather than whole organelles
Autophagic vacuoles → fuse with late endosomes or directly with active lysosomes
Starvation → need energy → autophagy increasing
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in rare cases, lysosomes enzymes exocytosis → extracellular digestion – Sperm : to penetrate the egg surface – Rheumatoid arthritis : release of lysosomal
enzymes into the joints • Cortisone and hydrocortisone (steroid hormone)
stabilized lysosomal membrane to inhibit enzyme release
Lysosome protein targeting
Mannose 6-phosphate (M6P) residues target soluble proteins to lysosomes
M6P receptor bind M6P → specific and tightly at acidic pH6.5, at trans-Golgi pH < 6 → bound lysosomal enzymes are released with late endosomes Phosphatase within late endosomes remove the phosphate from M6P on
lysosomal enzyme → prevent rebinding to the M6P Vesicle budding from last endosomes recycle the M6P receptor back the trans-
Golgi
The acid hydrolases in the lysosome are sorted in the TGN based on the chemical marker mannose 6-phosphate.
lysosomal enzymes (e.g., acid hydrolases) possess N- linked oligosaccharide as sorting signal
Trans-Golgi and cell surface → soluble protein → lysosomal enzymes
endocytosis
Cargo: mannose 6Cargo: mannose 6-- phosphatephosphate--tagged tagged lysosomallysosomal hydrolaseshydrolases
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• Mannose-6-phosphate receptors (MPRs) – In the interior surface of the TGN (pH 6.4) – Favor binding of lysosomal enzymes to
receptors → and then, packing into clathrin- coated transport vesicles → endosome
• In animal cells – Lysosomal enzymes: TGN → early endosomes → late endosomes → pH 5.5 → lysosomal enzymes to dissociate from the MPRs
– Receptors is recycled to TGN
I-cell disease: human genetic disorder
• Defective phosphotransferase • Absence of mannose-6-phosphate • Lysosomal enzymes were released to cell
Two secretory types: from trans Golgi to cell surface Constitutive secretion Regulated secretion: pancreatic β cell regulated by glucose, release
insulin No shared sorting sequence is found. Protein aggregation is observed in trans Golgi network, buds from
trans Golgi, & regulated secretory vesicles. Regulated secretory vesicles contain 3 proteins, chromogranin A,
chromogranin B, & secretogranin II, that form aggregates when incubated at pH 6.5 & 1 mM Ca2+.
Aggregates do not formed at the neural pH of ER.
Protein aggregation in trans-Golgi may function in sorting proteins to regulated secretory vesicles.
constitutive secretory proteins are sorted into transport vesicles at trans-Golgi network (TGN) – immediate movement to plasma membrane – release at PM via exocytosis
regulated secretory proteins are sorted into secretory vesicles at TGN – proteins are concentrated and stored until stimulus received to elicit exocytosis
nerve impulse hormonal stimulus
– ↑ [Ca+2] in cytoplasm needed to trigger fusion of vesicles with plasma membrane
sorting to lysosomes via late endosomal compartment – lysosomal enzymes – lysosomal membrane proteins
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Some membrane and secretory proteins initially are synthesized as long-lived, inactive protein → termed proproteins (soluble lysosomal enzyme aslo called proenzyme) → proteolytic → mature
Proteolytic conversion occur after the proprotein has been sorted in the trans Golgi vesicles
Mature vesicle
trans
Proteolytic processing of proproteins in constitutive and regulated secretion pathway
Several pathways sort membrane proteins to the apical or basolateral region of the polarized cells
Epithelial cells divided into apical and basolateral, has tight junction.
Tight junction prevent the movement of plasma membrane protein between different membrane. For different transport distribution.
The mechanism not well know. But they protein trafficking from trans Golgi.
GPI
MDCK cell (hepatocytes) are infected with VSV and influenza virus.
How are proteins efficiently and accurately targeted and maintained on the cell surface of polarized cells?
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Membrane trafficking is critical to Polarity • Sorting at the Tran
Golgi • Retention After
Secretion • Sorting After
Endocytosis • Sorting Signals
17.5 Receptor-mediated endocytosis and the sorting of internalized proteins
Phagocytosis: take up whole cell or large particle. non selective actin mediated process, extension of the membrane. marcophage
Pinocytosis: small droplets of extracellular fluid and any material dissolved , non-specifically
Receptor-mediated endocytosis: specific receptor involved.
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Transcytosis
In the infant intestine, antibodies are ingested from mother’s milk.
They bind to Fc receptors on the apical surface of the intestine.
The IgG-FcR complex is transcytosed to the basolateral side where the IgG is released.
The empty FcR is then transcytosed back to the apical side.
The pH values on either side of the
Polarized Epithelia Have Apical and Basolateral Specific Endosomes
• The additional complexity of the plasma membrane requires extra endosomal compartment s for sorting.
An epitope on apoB interacts with the LDL receptor on the cell surface. Each LDL contains 1500 molecules of cholesteryl esters.
epitope
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Uptake of low-density lipoproteins (LDL) is one of the best understood examples of receptor-mediated endocytosis.
LDL is a protein-lipid complex that transports cholesterol-fatty acid esters in the blood stream.
LDL normally supplies cholesterol to cells.
Defects in the endocytic process result in high blood levels of LDL.
High LDL predisposes individuals for atherosclerosis.

Endocytic pathway for internalizing LDL. pinocytosis
Normally, LDL binds the receptor and the receptors collect in coated pits through association with AP2 (adaptins) and clathrin.
Packed with cholesterol
LDL particles, water soluble carriers, transport cholesterol LDL receptors bind LDL particle via Apo-B, undergo endocytosis
and are transported to late endosome compartment LDL receptors are recycled back to cell surface LDL particles are sorted into transport vesicle & targeted to
lysosomes in lysosomal compartment, lysosomal hydrolases convert: – apo-B → amino acids – cholesterol esters → cholesterol + fatty acids
phospholipids, triglycerides
cell membranes, lipid droplets, steroid hormones, bile acids
receptor mutants have been useful in discovering various sorting signal motifs
one LDL receptor mutant, but can bound LDL normally; however,ligand-receptor complexes failed to
internalize and to cluster in clathrin coated pits Tyr → Cys mutation in cytosolic domain, which is within Tyr-X-X-φ motif and unable to
bind μ2 of AP2 heterotetramer complex general sorting signal motifs should be used as a CLUE, not a fact
LDL-receptor did interact with clathrin/AP2 formed complex
Some hypercholesterolemia
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Acidic pH of late endosomes causes most receptor-ligand complexes to dissociate.
Normally, LDL binds the receptor and the receptors collect in coated pits through association with adaptins and clathrin.
Some individuals have defects in the cytoplasmic domain recognized by adaptin so the receptors never collect in the coated pits.
Other genetic defects that result in elevated blood levels of LDL:
•absence of LDL receptor. •defective LDL-binding site in the LDL receptor.
Studies of familial hypercholesterolemia
Lead to discovery of LDL receptor & mechanism of receptor- mediated endocytosis.
Mutant LDL receptors -> identify NPXY sorting signal that binds to a subunit of AP2 complex, which is also mutated in some patients.
Mutational studies of other receptors YXXF sorting signal LL sorting signal
Model for pH-dependent binding of LDL by LDL receptor.
7 repeat (R1-R7) in the ligand-binding domain.
R4 and R5 most critical for LDL binding. Histidine rich in propeller() → acid
condition → positively charged propeller → high affinity to ligand binding arm (negative) → release LDL particle
A conserved set of GTPase switch proteins controls assembly of different vesicle coats.
A conserved set of GTPase switch proteins controls assembly of different vesicle coats.
Monomeric Rab-GTPases
Clathrin-coated vesicles
Lysosomes isolate digestive enzymes from the rest of the cell
Lysosomes develop form endososomes
Autophagy: The original recycling system
Extracellular digestion
Polarized Epithelia Have Apical and Basolateral Specific Endosomes
Acidic pH of late endosomes causes most receptor-ligand complexes to dissociate.
Studies of familial hypercholesterolemia