Chapter 17: Vesicular traffic, secretion, and endocytosis

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1 Chapter 17: Vesicular traffic, secretion, and endocytosis SEM of the formation of clathrin-coated vesicles on the cytosolic face of the plasma membrane Outline: 1. Techniques for studying the secretory pathway 2. Molecular mechanisms of vesicular traffic 3. Vesicular trafficking in the early stages of the secretory pathways 4. Protein sorting and processing in late stages of the secretory pathways 5. Receptor mediated endocytosis and the sorting of internalized proteins 6. Synaptic vesicle function and formation 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 Overview of secretory & endocytic pathways: Transport vesicles 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 17.1 Techniques for studying the secretory pathway: Pulse-chase labeling & EM autoradiography Tissue sections of pancreas acinar cells -> a brief incubation (3 min) with H 3 -Leucine -> transfer to unlabeled medium & incubate for a period of time (0, 7, 37, 117 min) -> cover tissue sections with photographic emulsion - > EM

2 17.1 Techniques for studying the secretory pathway: Use of temperature-sensitive mutant proteins (e.g. vesicular stomatitis virus 水疱口炎病毒 VSV G protein) At restrictive temp. of 40 o C, newly made G protein is misfolded & retained within ER. At permissive temp. of 32 o C, 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 min 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 17.1 Techniques for studying the secretory pathway: Use temperature-sensitive mutant, VSVG-GFP. 40oC the protein in ER 32oC move Golgi plasma membrane 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 Form ER to Golgi about 60min 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

3 Addition & processing of N-linked oligosaccharides in R-ER of vertebrate cells Add Remove 2 mannose Remove 3 mannose In cis, specific glycosidase Cleavage by endoglycosidase D. glycosidases (cis-) endoglycosidase D Fig17-3 Processing of N-linked oligosaccharide chains on glycoproteins within cis-, medial-, and trans-golgi cisternae in vertebrate cells 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. 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 Protein folding ok move golgi can cleavage

4 Yeast sec (secretion) mutants protein 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 Fig 17-5 Phenotypes of yeast sec mutants identified stages in the secretory pathway 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 Cell-free transport assay To plasma membrane Without energy Can not add Fig 17-6 Protein transport from Golgi cisternae to another can be assayed in a cellfree system Protein need modification in Golgi Proof: golgi can retrograde vesicular transport for midification Normal expression

5 Tradional Model - Golgi is a static organelle. Secretory proteins move forward in small vesicles. Golgi resident proteins stay where they are. Two Models For Cis to Trans-Golgi Progression 17.2 Molecular mechanisms of vesicular traffic Vesicle transport: from organelle (Donor) target organelle (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 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. Fig 17-7 Overview of vesicle budding and fusion with target membrane 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 retrograde ARF (ADP Ribosylation Factor)

6 Different coated proteins Clathrin and adapter protein (AP): vesicles transport proteins from the plasma membrane and trans-golgi network to late endosomes With AP1: Golgi to endosome 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) 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 AP: complex consists of four different subunits 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 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 ARF (ADP Ribosylation Factor) protein exchanges bound GDP for GTP and then binds to its receptor on Golgi membrane

7 Monomeric GTPase control coat assembly Specific receptor Cargo protein 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 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 by charperone (hsp70) 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 Major coat protein: clathrin & adaptin There are at least four types of adaptins, each specific for a different set of cargo receptor.

8 Coated vesicles accumulate during in vitro budding reactions in the presence of a nonhydrolyzable analog of GTP Targeting sequence on cargo proteins make specific molecular contacts with coat protein Golgi membrane + COPI coat proteins and GTP bud off Non-hydrolyzable GTP prevent disassembly of the coat after vesicle release The retrieval pathway to the ER uses sorting signals Lys-Asp-Glu-Leu (KDEL) Short retrieval signal at c-terminal Resident ER membrane protein KKXX at c-terminal end direct interact with COPI coat Different Rab GTPases & Rab effectors control docking of different vesicles on target membranes: vesicle docking controlled by Rab protein. Vesicle docking controlled by Rab proteins 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

9 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. Dissociation of SNARE complexes after membrane fusion is driven by ATP hydrolysis. 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) t-snares are syntaxin SNAP-25 attached to membrane by hydorphobic anchor. Formation of four-helix bundle: But, VAMP in COPII (1), Syntaxin with cis, each (1) and SNAP- SNARE 25 (2) has provide one helix SNARE complex had specificity 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 (NEMsensitive factor, blocked by N-ethylmaleimide) & α- SNAP (soluble NSF attachment protein). hexamer

10 Soluble (i.e. cytoplasmic) Factors 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 Synaptobrevin Identified and cloned ~ Originally called VAMP (Vesicle-Associated Membrane Protein) and sometimes abbreviated as Syb Cleaved by tetanus toxin (failure of exocytosis = death) Spans vesicle membrane ~ 13 kda Inject antibodies to Synaptobrevin and release is blocked Dissociation of SNARE complexes after membrane fusion is driven by ATP hydrolysis. Rizo and Sudhof 2002 Nature Rev. Neurosci. ATP is not actually required for release once vesicles are docked, but is thought to break down the SNARE complexes to promote recycling.

11 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

12 Summary Conformational changes in viral envelope proteins trigger membrane fusion. Proteins moved between organelles of secretory pathway fully folded, enclosed in vesicles -proteins only have to cross ER membrane Large amount of vesicular traffic between ER, Golgi, lysosomes and plasma membrane Vesicle budding is function of protein coats Cargo selected by sorting/cargo receptors Specificity of fusion controlled by Rabproteins, v-snares and t- SNAREs Three HA1 and three HA2 HA1 The structure of influenza hemagglutinin (HA) 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. 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. Binding of fusion peptide to HA2 disrupted. Globular domains dissociate. Loop segment forms a continuous helix. Fusion peptide inserts into endosomal membrane.

13 Viral fusion proteins and SNAREs may use similar strategies Model for membrane fusion directed by hemagglutinin (HA) 17.3 Early stages of the secretory pathway 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

14 Vesicle-mediated protein trafficking between ER & cis-golgi Anterograde-COPII vesicle Retrograde-COPI vesicle Cargo protein vsnares (yellow) Rab important Fig Vesicle-mediated protein trafficking between the ER and cis-golgi COPII vesicles mediate transport from the ER to the Golgi Formation of COPII vesicles: triggered cytosol by Sec12 induced catalyzes the ER lumen 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). 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. 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. 3-D structure of ternary complex comprising the COPII coat proteins (Sec23, Sec24) & Sar1-GTP. ph probably alters KDEL receptor conformation - regulating cargo binding and inclusion in COPI vesicles. PH high

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 Anterograde transport through the Golgi occurs by cisternal progression Cisternal progression: protein form cis to trans Trans more large than cis Lys-Lys-X-X in KDEL receptor or membrane receptor( Retrieval of ERresident 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. Large macromolecular assemblies (e.g. algal scales & precollagen aggregates) are too large and never found in transport vesicles. COP II anterograde transport rough ER Golgi COP I retrograde transport between Golgi stacks retrograde transport cis-golgi rough ER clathrin PM late endosome (e.g., endocytosis) 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) 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 ( 三曲線 ) 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. Structure of clathrin coats (36 triskelions) Clathrin-coated vesicles Types of AP complexes: AP1, AP2, AP3, GGA. Clathrin/AP (adaptor protein complex) Clathrin/GGA1/2/3 GGA identified in GGAs in human, 2 in yeast Tyr-X-X-φ signal sequence (φ hydrophobic), from cis-golgi budding; interac with AP1 GGA adaptin complexes recognize specific cargo and link it to clathrin assembly

17 GGAs Golgi-associated, γ-adaptin ear homologous, ARF-binding proteins GGAs are clathrin adaptors Localized to the trans-golgi network (TGN) Transport to the endosome/lysosome system AP-1 GGA Hinge Nakayama et al., Cell Structure and Function 28: (2003) GGA domains and function VHS: cargo receptor binding and membrane targeting GAT: ARF-interacting and Golgi localization Hinge: clathrin-binding GAE: binding accessory proteins mannose 6- phosphate receptor COPI model Comparison between models ARF recruits coat proteins ARF hydrolysis is inhibited by cargo receptor COPI associates with membrane through ARF COPI promotes ARF hydrolysis ARF hydrolysis drives coat protein dissociation GGA model ARF recruits GGA which binds clathrin GGA inhibits ARF hydrolysis GGA has a docking site via M6PR ARF dissociates before cargo loading ARF does not help coat disassembly Robinson et al., Current Opinion in Cell biology 13: (2001)

18 Dynamin is required for pinching off ( 脫離 ) of clathrin vesicles Dynamin is needed for left donor membrane, need GTP hydorlysis COPI and II did not need No GTP hydrolysis no pinching off of clathrin-coated vesicles Model for dynamin-mediated pinching off of clathrin/ap-coated vesicles Fig GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles in cell free extract (GTP-γ-S) Lysosomes and cellular digestion Containing digestive enzyme Lipids, carbohydrates, nucleic acids, proteins, extracellular 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 ( ) 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

19 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 Two ways By ATP-dependent proton pump (late endosomal lumen to ) 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

20 Extracellular digestion Lysosome protein targeting 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 Mannose 6-phosphate (M6P) residues target soluble proteins to lysosomes Trans-Golgi and cell surface soluble protein lysosomal enzymes endocytosis Cargo receptor: mannose 6-phosphate 6 receptor Cargo: mannose 6-6 phosphate-tagged tagged lysosomal hydrolases lysosomal enzymes (e.g., acid hydrolases) possess N- linked oligosaccharide as sorting signal The acid hydrolases in the lysosome are sorted in the TGN based on the chemical marker mannose 6-phosphate. 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 COPI and II

21 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 clathrincoated 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 Protein aggregation in trans-golgi may function in sorting proteins to regulated secretory vesicles. 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. 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

22 Some proteins undergo proteolytic processing after leaving the trans-golgi 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 Proteolytic processing of proproteins in constitutive and regulated secretion pathway Mature vesicle immature trans Many proinsulin in immature vesicle Much insulin in mature vesicle How are proteins efficiently and accurately targeted and maintained on the cell surface of polarized cells? 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 GPI different transport distribution. The mechanism not well know. But they protein trafficking from trans Golgi. MDCK cell (hepatocytes) are infected with VSV and influenza virus.

23 Membrane trafficking is critical to Polarity Sorting at the Tran Golgi Retention After Secretion Sorting After Endocytosis Sorting Signals Basolateral: Tyrosine or DiLeucine Apical: N or O-linked Glycosylation Or TM domain Three Destinations After Endocytosis In a Polarized Cell 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.

24 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. Polarized Epithelia Have Apical and Basolateral Specific Endosomes The additional complexity of the plasma membrane requires extra endosomal compartment s for sorting. The ph values on either side of the epitope An epitope on apob interacts with the LDL receptor on the cell surface. Each LDL contains 1500 molecules of cholesteryl esters.

25 Endocytic pathway for internalizing LDL. pinocytosis 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. Normally, LDL binds the receptor and the receptors collect in coated pits through association with AP2 (adaptins) and clathrin. Packed with cholesterol endocytosis uncoating Fusion with endosome 突出 Free cholesterol ph 5 induces dissociation 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 Some hypercholesterolemia 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 cell membranes, lipid droplets, steroid hormones, bile acids phospholipids, triglycerides LDL-receptor did interact with clathrin/ap2 formed complex

26 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 Model for ph-dependent binding of LDL by LDL receptor. Lead to discovery of LDL receptor & mechanism of receptormediated 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 7 repeat (R1-R7) in the ligand-binding domain. R4 and R5 most critical for LDL binding. Histidine rich in propeller( 螺旋槳 ) acid condition positively charged propeller high affinity to ligand binding arm (negative) release LDL particle