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Cell membrane. Lecture-8: Vesicular traffic (II). Reference: Chapter 14 Lodish Harvey et al. (2008) Molecular Cell Biology (6 th edition) Publisher: W.H. Freeman and Company. Exocytosis. Constitutive and regulated secretion.

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Lecture-8: Vesicular traffic (II)


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    1. Cell membrane Lecture-8: Vesicular traffic (II) Reference: Chapter 14 Lodish Harvey et al. (2008) Molecular Cell Biology (6th edition) Publisher: W.H. Freeman and Company

    2. Exocytosis

    3. Constitutive and regulated secretion

    4. Lysosomes are small bodies, enclosed by membranes, that contain hydrolytic enzymes in eukaryotic cells. 25.7 Protein localization depends on further signals

    5. 25.7 Protein localization depends on further signals Figure 25.22 A transport signal in a trans- membrane cargo protein interacts with an adaptor protein.

    6. Figure 25.23 A transport signal in a luminal cargo protein interacts with a transmembrane receptor that interacts with an adaptor protein. 25.7 Protein localization depends on further signals

    7. Insulin is a good example of a protein that is stored in secretory vesicles until a cell receives an signal to secrete the insulin. Removal of the Pre-sequence (not shown), folding and disulfide bond formation occur in ER. Processing to the final form occurs in the secretory vesicle. This is an example of a protein that you would not want to treat with mercaptoethanol because reduction of disulfide bonds would inactivate the protein.

    8. “pre-pro-proteins” Some proteins are processed in secretory vesicles into multiple small polypeptides. One explanation for this approach is that the small polypeptides are too short to be cotranslationally transported into the ER.

    9. Figure 25.5 Processing for a complex oligosaccharide occurs in the Golgi and trims the original preformed unit to the inner core consisting of 2 N-acetyl-glucosamine and 3 mannose residues. Then further sugars can be added, in the order in which the transfer enzymes are encountered, to generate a terminal region containing N-acetyl-glucosamine, galactose, and sialic acid. 25.7 Protein localization depends on further signals

    10. Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments. 1. Sorting in TGN 2. Protection from protease digestion 3. Cell to cell adhesion via selectins

    11. 25.8 ER proteins are retrieved from the Golgi Figure 25.24 An (artificial) protein containing both lysosome and ER-targeting signals reveals a pathway for ER-localization. The protein becomes exposed to the first but not to the second of the enzymes that generates mannose-6-phosphate in the Golgi, after which the KDEL sequence causes it to be returned to the ER.

    12. 25.8 ER proteins are retrieved from the Golgi Figure 25.24 An (artificial) protein containing both lysosome and ER-targeting signals reveals a pathway for ER-localization. The protein becomes exposed to the first but not to the second of the enzymes that generates mannose-6-phosphate in the Golgi, after which the KDEL sequence causes it to be returned to the ER.

    13. Endocytosed molecules that are destined for the lysosome go from the early endosome to the multivesicular body to the late endosome. Fusion of transport vesicles carrying acid hydrolases from the Golgi causes the late endosome to mature into a lysosome.

    14. In some cases, both the receptor and the ligand are transported to the lysosome. This is the case for EGF and its receptor. EGF triggers a cell to proliferate but the signal is only required for a short time. To limit the response time both the receptor and the ligand are removed from the membrane.

    15. Mosaic organization of endosomes: subdomains

    16. Tubular-vesicular endosomes sort membrane components from lumenal components Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

    17. Experimental demonstration that internalized receptor-ligand complexes dissociate in endosomes Hepatocyte: Asialglycoproteins and their receptor. Sorting of membrane from contents: surface area to volume ratio. Narrow diameter tubules

    18. Late Endosomes Contain Internal Vesicles Maturation from early to late endosomes occurs through the formation of multivesicular bodies (MVBs). The MVBs move deeper into the cytoplasm fusing with each other and pre-exisiting late endosomes. These structures are characterized by the formation of internal vesicles. Vesicles inside of vesicles.

    19. Late Endosomes Sort By Selective Internalization of Limiting Membrane The formation of internal vesicles by pinching off of the limiting membrane of MVBs/late endosomes is a sorting process. Membrane proteins destined for degradation are marked with a covalent mono-ubiquitin tag. These mono-ubiquitinated membrane proteins are sorted into invaginating buds that pinch off into internal vesicles. Internal vesicles and their contents are degraded in the lysosome.

    20. The Machinery for MVB formation is used by retroviruses to bud 1. Ubiqutinated Hrs protein on the endosome recruits Ub-tagged TM cargo to buds then recruits ESCRT complexes. 2. ESCRT Required to pinch off internal vesicles. 3. The Vps4 ATPase disassembles ESCRT. 4. Ub-Gag mimics Hrs Recruiting ESCRT to HIV PM buds. 5. ESCRT pinches off buds Releasing free virus, and Vps4 Recycles ESCRT.

    21. HIV Budding from the cell surface

    22. Vesicle budding and fusion • Coated vesicles are formed by polymerization of coat proteins onto a membrane to form vesicle buds and then pinch off from the membrane to release a complete vesicle. • Vesicle budding is initiated by recruitment of a GTP-binding proteins: - ARF protein is for both COPI and clathrin vesicles. - Sar1 protein is for COPII vesicles. • Vesicles fuse with its target membrane in a process involves interaction of cognate SNARE proteins.

    23. Vesicle budding • Step 1: Soluble Sar1-GDP is converted to Sar1-GTP by Sec12, a GEF on ER membrane. Binding of GTP causes a conformational change in Sar1 that exposes its hydrophobic N-terminus, leading to the anchorage of Sar1 to the ER membrane. • Step 2: Attached Sar1-GTP serves as a binding site for the Sec23/Sec24 coat protein complex (COPII subunits). Membrane cargo proteins are recruited to the vesicle bud by binding of sorting signal sequence. • Step 3: Once vesicles are released, the Sec23 subunit promotes Sar1 GTPase activity and leads to GTP hydrolysis by Sar1. • Step 4: Release of Sar1-GDP from the vesicle membrane causes disassembly of the COPII coat.

    24. Sorting signals in cargo proteins • For membrane cargo proteins, the vesicle coat selects these proteins by directly binding to their cytoplasmic sorting signals on cytosolic portion, while for soluble luminal proteins, the vesicle coat selects these proteins by indirectly binding to their luminal sorting signalsthrough a cargo receptor.

    25. Regulation of endocytosis. Several different kinds of proteins and lipids regulate internalization and endosomal sorting. Rab proteins are membrane associated, Ras-like GTPases that control membrane fusion. Different Rabs are associated with particular endosomes. Inositol phospholipids (phosphoinositides) constitute a small fraction of the phospholipids in the plasma membrane and endosomal membranes. Distinct regions of the plasma membrane and different endosomes are enriched in particular varieties of phosphoinositides which bind with different affinities to proteins with lipid-binding domains. For example, the ENTH domain of Epsin (see below) binds PI(4,5)P2, which is enriched at the plasma membrane in vertebrate cells. Some transmembrane proteins have cytoplasmically located internalization signals that are part of their primary amino acid sequence, and these may bind AP-2. Alternatively, a ubiquitin (Ub) polypeptide that serves as an endocytosis signal may be added posttranslationally to the cytoplasmic domain, and these signals

    26. v-SNARE (VAMP) t-SNARE (SNAP-25) t-SNARE (Syntaxin) The SNARE complex • During exocytosis of secreted proteins, the v-SNARE is VAMP (vesicle- associated membrane protein). The t-SNAREs are syntaxin, an integral membrane protein, and SNAP-25 which is attached to membrane by a hydrophobic lipid anchor. • The four helices (one from VAMP, one from syntaxin, and two from SNAP-25) to coil around one another to form a four-helix bundle. The stability of bundle is hold by the electrostatic interactions of opposite- charged amino acids between helices. • The dissociation of SNARE complexes requires energy and two proteins, NSF (NEM-sensitive factor) and α-SNAP (soluble NSF attachment protein). NSF associates with a SNARE complex with the aid of α-SNAP, which hydrolyzes ATP and releases energy to dissociate SNARE complex.

    27. Vesicles ducking and fusion • Step 1: The ducking between the vesicle and the target membrane is mediated by the interaction between the vesicle-attached Rab GTPase and its effector on the target membrane. • Step 2: VAMP proteins on the vesicle surface interact with the cytosolic domains of syntaxin and SNAP-25 on the target membrane to form a coiled-coil SNARE complex, which brings two membranes close together. • Step 3: Membrane fusion immediately after the formation of SNARE complex. • Step 4: NSF associating with α-SNAP binds to the SNARE complexes. The NSF-catalyzed hydrolysis of ATP then drives disassembly of the SNARE complexes. At the same time, Rab-GTP is hydrolyzed to Rab-GDP and dissociates from the Rab effector.

    28. Vesicle trafficking between ER and cis-Golgi • Step 1-3: the anterograde transport from the ER to cis-Golgi is mediated by COPII vesicles. These vesicles contain newly synthesized proteins destined for the Golgi, cell surface or lysosome. • Step 4-6: the retrograde transport from the cis-Golgi to ER is mediated by COPI vesicles. The purpose of this transport is to retrieve v-SNAREs, membranes and misfolded proteins back to the ER.

    29. KDEL receptor in retrograde transport • Most soluble ER-resident proteins carry a Lys-Asp-Glu-Leu (KDEL) sequence at their C-terminus, forming KDEL sorting signal. • The KDEL sorting signal is recognized and bound by the KDEL receptor which is located mainly in the cis-Golgi and in both COPII and COPI vesicles. • The binding affinity of KDEL receptor is enhanced at low pH. Thus, the difference in the pH of the ER and Golgi favors binding of KDEL-bearing proteins to the receptor in Golgi-derived vesicles and their release in the ER. • This retrieval system prevents depletion of ER luminal proteins such as chaperone proteins.

    30. Models for the polarization of the Golgi • In the vesicular transport model, the Golgi cisternae are static organelles, which contain their resident proteins. The passing of molecules from cis to trans through anterograde transport. • In the cisternal maturation model, the Golgi cisternae are dynamic organelles. Each cisterna matures as it migrates forward. At each stage, the Golgi-resident proteins carried forward in a cisterna are moved backward to an earlier compartment by retrograde transport.

    31. Tight junctions divide the PM of polarized cells into domains • Apicobasal Polarity is associated with many cell-types. • Epithelial cells form ion-tight monolayers of high electrical resistance. • Apical and Basolateral Domains are different in Lipid and Protein Composition

    32. Polarized Cells

    33. Membrane trafficking is critical to Polarity • Sorting at the Trans-Golgi • Retention After Secretion • Sorting After Endocytosis • Sorting Signals Basolateral: Tyrosine or DiLeucine Apical: N or O-linked Glycosylation Or TM domain

    34. Three Destinations After Endocytosis In a Polarized Cell

    35. Polarized Epithelia Have Apical and Basolateral Specific Endosomes • The additional complexity of the plasma membrane requires extra endosomal compartments for sorting.

    36. Basolateral Targeting and Human Disease Koivisto et al., 2001: In the familial hypercholesterolemia (FH)-Turku LDL receptor allele, a mutation of glycine 823 residue affects the signal required for basolateral targeting in MDCK cells. We show that the mutant receptor is mistargeted to the apical surface in both MDCK and hepatic epithelial cells, resulting in reduced endocytosis of LDL from the basolateral/sinusoidal surface. This work suggests that a defect in polarized LDL receptor expression in hepatocytes underlies the hypercholesterolemia in patients harboring this allele.

    37. Processing of N-linked glycosylation in Golgi • The Golgi complex is organized into 3-4 cisternae, which contain different enzymes for protein glycosylation. • N-linked glycosylation in the Golgi: > In cis-Golgi, three mannose residues are removed (1). > In medial-Golgi, three GlcNAc (2,4) • and one fucose (5) residues are added, • while two mannose (3) residues are • removed. • > In trans-Golgi, three galactose (6) • residues are added, followed by the • linkage of N-Acetylneuraminic acid (7) • on each galactose residue. • Each enzyme move dynamically from the later to the earlier cisterna through retrograde vesicle transports. (GlcNAc)

    38. Evidence of Golgi cisternal maturation  Yeast cells expressing: > the cis-Golgi protein Vrg4-GFP (green) > the trans-Golgi protein Sec7-DsRed (red) • A compartment rarely contains both cis- and trans-Golgi proteins at the same time.

    39. Endocytosis Why do cells need endocytosis? Is there more than one endocytic pathway ? Clathrin-mediated uptake Caveolae Non-clathrin/non-caveolae pathways Pinocytosis/ Phagocytosis What are the functional consequences of endocytosis? How are endocytic structures formed and how do they know where to go? Where do the textbook models come from?

    40. Is cholera toxin internalized to the Golgi complex by a clathrin-dependent process? Epsin and eps15 mutants inhibit clathrin-mediated transferrin (Tf) uptake to recycling endosomes Epsin and eps15 mutants do not affect cholera toxin B-subunit (CTXB) uptake to the Golgi complex (marked by b-COP) Suggests CTXB is delivered to the Golgi complex by a clathrin-independent pathway b-COP: Marker for the Golgi complex

    41. Does internalized CTXB pass through early endosomes? Early endosome function requires the GTPase Rab5 Dominant negative rab5 S34N (GDP bound) expression perturbs early endosomes and blocks transferrin uptake Rab5 S34N does not affect delivery of CTXB to the Golgi complex Suggests CTXB does not pass through early endosomes Nichols et al. 2001 J. Cell Biol.

    42. Active Membrane Transport – Review

    43. The endocytic pathway is divided into the early endosomes and late endosomes pathway. • Materials in the early endosomes are sorted: • Integral membrane proteins are shipped back to the membrane; • Other dissolved materials and bound ligands Multivesicular body (MT mediated transport) the late endosomes. • Dissociation of internalized ligand-receptor complexs in the late endosomes. Molecules that reach the late endosomes are moved to lysosomes.

    44. The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy.

    45. lysosomes • Lysosomes contain about 40 types of hydrolytic enzymes. For optimal activity, they need to be activated by proteolytic cleavage and an acidic environment, which is established by the V-class H+ pumps on lysosomal membrane. • Mature endosomes containing numerous vesicles in their interior are usually called multivesicular endosomes. Fusion of a multivesicular endosome directly with a lysosome releases the internal vesicles into the lumen of the lysosome, where they can be degraded. • Lysosomal membrane proteins are not incorporated into internal endosomal vesicles, thus keeping them away from degradation.