MB207 Molecular Cell Biology

1. MB207 Molecular Cell Biology Intracellular Vesicular Traffic • Outline: • Principles of vesicular traffic • Transport from ER through Golgi Apparatus • Transport from trans Golgi network to lysosomes • Transport into cell from plasma membrane: endocytosis • Transport from trans Golgi network to the cell exterior: exocytosis

2. Endocytosis and exocytosis

3. Vesicular Transport • Process in which membrane-enclosed transport vesicles transport proteins from one membrane-enclosed compartment to another. • Shape: spherical, larger irregular-shaped vesicles. • Proteins do not move across the lipid bilayer of any membranes. But only move between topologically equivalent compartments (eg. Lumen of ER to lumen of Golgi to exterior of the cell). Principles of vesicular transport • A protein-coated membrane-enclosed transport vesicle buds off from the membrane of donor compartment carrying a variety of specifically selected cargo molecules. • Transport vesicle binds to the target compartment and fuse with the membrane of the target compartment. • Cargo molecules transfer into lumen of the target compartment and inserting the vesicular membrane components into the target compartment membrane. • Overview of vesicle transport: • Budding • Uncoating • Transport • Docking • Fusion

4. Question 1: How do transport vesicles form? (Budding) Question 2: What deforms the membrane to cause a vesicle form? (A planar phospholipid lipid bilayer wants to remain flat. But, the small transport vesicles that are seen in cells are small and highly curved. It is this protein coat that causes the membrane to deform and form a transport vesicle.) Coat proteins assemble on the membrane surface and curve the membrane into a vesicle

5. exchange of membrane material and vesicular lumenal contents, each organelle maintains its own highly specialized characteristics.

8. Three types of protein-coated vesicles

9. 3 main types of coated vesicles (each type use for different transport steps in the cell) • COP II-coated (Sec complex) • Move material from ER to the cis-Golgi complex (5 protein complex, including SARI GTPase) • COP I-coated (7 different COP subunits) • Retrograde movement from Golgi cisternae to ER (8 protein complex, including ARF GTPase) • Clathrin-coated (Clathrin + adaptin) • Move from the TGN (trans Golgi network) to endosome • Move from plasma membrane (endocytosis) to endosome • Golgi to lysosome

10. Clathrin-coated vesicles • Major proteins in clathrin-coated vesicle are clathrin and adaptin • Each clathrin subunit composes of heavy and light chain • Three clathrin subunits form a three-legged structure called triskelion. • Adaptin bind to both clathrin and transmembrane cargo receptor, which in turn bind to the target cargo • As coated bud grows, a GTP-binding –protein, dynamin, assemble around the neck of each bud. Hydrolysis of GTP will regulate the speed of the pinching-off process for membrane fusion. • Shortly after vesicle formed, energyrequired to remove clathrin coat Clathrin triskelion ( 3 legged structure)

11. The assembly and disassembly of a clathrin coat • Curvature was introduced into the membrane which leads to the formation of uniformly sized coated buds. • The adaptins bind both clathrin trikelions and membrane-bound cargo receptors, thereby mediating the selective recruitment of both membrane and cargo molecules into the vesicle. • The pinching-off of the bud to form a vesicle involves membrane fusion, facilitated by the GTP-binding protein dynamin which assembles around the neck of the bud. • The coat of clathrin-coated vesicles is rapidly removed shortly after the vesicle forms.

12. Small GTPases regulate budding • 2 conformational states: • GDP (off) • GTP (on) • SarI proteins is a coat recruitment GTPase. • Inactive, soluble SarI-GDP binds to a GEF (called SecI2) in the ER membrane, causing SarI to release its GDP and bind to GTP. • A GTP-triggered conformational change in SarI exposes its fatty acid chain, which inserts into the ER membrane. • Membrane-bound, active SarI-GTP recruits COPII subunits to the membrane. This causes the membrane to form a bud which includes selected membrane proteins. • A subsequent membrane-fusion event pinches off and releases the coated vesicle.

13. Transport vesicle docking • Specificity in targeting ensures that membrane traffic proceeds in an orderly way. • Therefore, transport vesicles must be highly selective in recognizing the correct target membrane with which to fuse. • → transport vesicles display surface markers that identify them according to their origin and type of cargo while target membrane display complementary receptors that recognize the appropriate markers. • The recognition step is thought to be controlled mainly by two classes of proteins: • i) SNAREs (Soluble NSF Attachment Protein Receptor) • - central role both in providing specificity and in catalyzing the fusion of • vesicles with the target membrane. • ii) Rabs (GTPase) • - work together with other proteins to regulate the initial docking and • tethering of the vesicle to the target membrane.

14. SNAREs • compartment identifiers - required for vesicle docking and fusion • transmembrane protein that exist as complementary sets to ensure correct vesicle targeting: • i) vesicle membrane SNAREs, v-SNAREs • ii) target membrane SNAREs, t-SNAREs • Complementary sets of v-SNAREs and t-SNAREs contribute to the selectivity of transport-vesicle docking and fusion • The v-SNAREs are packaged together with the coat proteins during the budding of transport vesicles from the donor membrane and bind to complementary t-SNAREs in the target membrane. • After fusion, the v- and t-SNAREs remain associated in a tight complex. • The complexes have to be dissociated before the t-SNAREs can accept a new vesicle or the v-SNAREs can be recycled to the donor compartment for participation in a new round of vesicular transport. • Different v-SNAREs can be packaged with different cargo molecules when leaving the donor compartment.

15. Dissociation of SNARE pairs • Complexes have to be disassembled before the SNAREs can mediate new rounds of transport. • NSF protein cycles between membranes and the cytosol and catalyzes the disassembly process. NSF (an ATPase) uses the energy of ATP hydrolysis to solubilize and facilitates refolding of denatured proteins. • After the v-SNAREs have mediated the fusion of a vesicle on a target membrane, the NSF binds to the SNARE complex via adaptor proteins and hydrolyzes ATP to interfere the SNAREs apart.

16. Coiled-coil SNARE complexes • This complex are formed hold the vesicle close to the target membrane. Numerous noncovalent interactions between SNARE protein stabilize the coiled-coil structure. • The SNAREs responsible for docking synaptic vesicles at the plasma membrane of nerve terminal consists of three proteins. • The v-synaptobrevin and t-SNARE syntaxin are both transmembrane proteins and each contributes one α-helix to the complex. The t-SNARE Snap25 is a peripheral membrane protein that contributes two α-helices to the four-helix bundle. • Trans-SNARE complexes always consists of four tightly intertwined α-helices, three contributed by t-SNARE and one by a v-SNARE.

17. Rab protein: Docking of transport vesicle • Rab proteins are monomeric GTPases. • Rab proteins facilitate and regulate the rate of vesicle docking and the matching of v-SNAREs and t-SNAREs. • Rab proteins cycle between a membrane and the cytosol. In their GDP-bound state, they are inactive. They are active in the cytosol and in their GTP-bound state . • Rab protein hops onto the vesicle during budding. • SNARE proteins are tightly “capped” or turned off by association with other proteins. • Rab protein are specifically distributed through the secretory pathway.

18. Subcellular locations of some Rab proteins

19. Fusion of membranes mediated by SNAREs Tight SNARE pairing forces lipid bilayers into close apposition so that H2O are expelled from the interface. Lipid of 2 interaction leaflets of the barriers then flow between the membrane to form a connecting stalk. Lipid of the other 2 leaflets then contact each other forming a new bilayer, which widens the fusion zone (hemifusion). Split of the new bilayer completes the fusion reaction.

20. Entry of enveloped viruses into cells: HIV

21. Transport from the ER through the Golgi apparatus • Transport process from ER to Golgi apparatus and from Golgi apparatus to the cell surface and elsewhere. → the proteins pass through a series of compartments where they are successively modified. • Transport vesicles select cargo molecules and move them to the next compartment in the pathway, while others retrieve escaped proteins and return them to the previous compartment where they normally function. • Golgi apparatus → major site of carbohydrate synthesis as well as sorting and dispatching station for the products of ER. • Most proteins and lipids (acquired their appropriate oligosaccharides in the Golgi apparatus) are recognized in other ways for targeting into the transport vesicles going to other destinations.

22. The recruitment of cargo molecules into ER transport vesicles • By binding to the COPII coat, membrane and cargo proteins become concentrated in the transport vesicles as they leave the ER. • Membrane proteins are packaged into budding transport vesicles through the interactions of exit signals on their cytosolic tails with the COPII coat. • Some of the membrane proteins trapped by the coat in turn function as cargo receptors, binding soluble proteins in the lumen and helping to package them into vesicles. • Unfolded or imcompletely assembled proteins are bound to chaperones and are thereby retained in the ER compartment.

23. Protein leave ER in COPIl -coated transport vesicles • Vesicular tubular clusters are the structures formed when ER-derived vesicles fuse with one another. The clusters are relatively short-lived because they quickly move along microtubules to the Golgi apparatus. • COPII-coated vesicles leave the ER, uncoat and begin to fuse with one-another to form vesicular-tubular clusters. • These clusters associate with motor proteins that drag them along microtubules in an ATP-dependent process. • Meanwhile, retrograde transport removes certain components, purifying and concentrating the secretory cargo further.

24. Retrieval of ER resident proteins is receptor-mediated • Escaped ER resident proteins are retrieved from the Golgi by KDEL receptors that recognize specific retrieval signals in ER proteins. • The KDEL receptor present in the vesicular tubular clusters and the Golgi apparatus, captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER, • Upon binding its ligands in this low pH environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPI-coated vesicles.

25. A model for the retrieval of ER resident proteins • The retrieval of ER proteins begins in vesicular tubular cluster and continues from all environment of the ER, the ER resident proteins dissociate from the KDEL receptor, which is returned to the Golgi apparatus for reuse. • 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.

26. Golgi apparatus • The Golgi complex is typically disc-shaped with a stack of 4 - 6 cisternae. • Individual stacks may be interconnected in a large complex. • Golgi complex processes protein (glycosylation & sulfation) and synthesizes some polysaccharides. • Functionally distinct compartments, arranged from cis face (closest to ER) to trans (exit) face • Cis-Golgi network: sorts new proteins, separating those for return to the ER from those to pass to the next Golgi station • Trans-Golgi network: sorts proteins into vesicles bound for specific destinations i.e. lysosomes, secretory vesicles or the cell surface.

27. Functional compartmentalization of the Golgi apparatus • Post-translational modifications in the Golgi • Specific modifications occur in specific subcompartments, because modifying enzyme localization is tightly controlled. • The state of protein modification can identify how far a protein has proceeded in transport.

28. Two models for movement

29. Two Models For Cis to Trans-Golgi Progression Traditional 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.

30. Functions of Golgi apparatus • Sorting and processing of proteins and lipids coming from ER • Example mannose 6-phosphate is added to proteins that are destined for lysosomes 2. Oligosaccharide synthesis • Many protein from ER are glycosylated during their transit through the Golgi • Pectin and hemicellulose for the cell wall of plants; proteoglycans for the extracellular matrix in animal.

31. Transport from trans Golgi network to lysosomes • Lysosomes are organelles filled with hydrolytic enzymes that are used for controlled intracellular digestion of macromolecules • Turnover of cell’s macromolecules • Extracellular macromolecules from other cells • Bacteria (macrophages) • The interior of a lysosome is acidic (pH 4.6) • Unique enzymes of lysosomes are adapted to low pH • Advantages: • Digestive reactions more rapid at low pH • Leaked lysosomal enzymes do not destroy the cell (pH 7 to 7.3) • Low pH is maintained by a ATPase • pH decreases from ER to Golgi to endosome

32. Three pathways to degradation in lysosomes • Lysosome can be small (25 to 50 nm) or large (over 1 µm). • Multiple paths to deliver material to lysosomes. • Each pathway leads to intracellular digestion of materials derived from different source: • i) Through Late endosome - Developed from endosomes, an endocytotic vesicle derived from the plasma membrane. • ii) Through Phagosome - A vacuole formed around a particle absorbed by phagocytosis by the fusion of the cell membrane around the particle. • iii) Through Autophagosome - A membrane-bound body occurring inside a cell and containing decomposing cell organelles. • Materials delivered to lysosome may be degraded in the lysosome or expelled (exocytosis)

33. Transport of newly sythesized lysosomal hydrolases to lysosomes • Lysosomal proteins are synthesized in the RER and transported to the Golgi complex, just like secreted proteins. However, enzymes in the Golgi recognize and tag lysosome-bound proteins by phosphorylating the mannose residues. • The mannose-6-phosphate groups are recognized by the mannose-6-phosphate receptors (MPR) at the trans Golgi network and hence the recognition signals for packaging. • At late endosome / lysosome, the protein will dissociate from the receptors and the receptors can then be recycled.

34. Endocytosis: Transport into the cell from the plasma membrane • Internalization of external material including proteins located on the plasma membrane • Purpose: Cellular uptake of macromolecules usually en route to the lysosome. This could include the ingestion of metabolites, lipids like cholesterol through LDL and the LDL receptor or iron via the transferring receptor. Other reasons for endocytosis include the termination of cell surface events (i.e. signaling). • Types of endocytosis: • Phagocytosis or “cellular eating”: Ingestion of large particles such as microorganisms or dead cells (usually >250 nm in diameter) by phagocytes such as macrophages & neutrophils. • Pinocytosis or “cellular drinking”: Fluid-phase endocytosis primary for the uptake of fluids and solutes (~100 nm in diameter).

35. Mechanisms of Endocytosis • Similar to vesicle budding from the ER. • Coat protein clathrin is primarily responsible for the majority of vesicular traffic between the trans-golgi network and plasma membrane in both directions. • Another type of vesicles called caveolaecan also be used for pinocytosis. Caveolae collect cargo by the lipid composition of the caveolar membrane rather than the protein coat. Formation of clathrin coated vesicles from the plasma membrane

36. Endocytotic pathway from the plasma membrane to lysosomes • Maturation from early to late endosomes occurs through the formation of multivesicular bodies, which contain large amounts of invaginated membrane and internal vesicles. • These bodies move inward along microtubules, and recycling of components to the plasma membrane continues as the bodies move. • The multivesicular bodies gradually turn into late endosomes, either by fusing with each other or by fusing with pre-existing late endosomes. • The late endosomes no longer send vesicles to the plasma membrane but communicate with the trans Golgi network via transport vesicles, which deliver the proteins that will convert the late endosome into a lysosome.

37. Receptor-mediated endocytosis & fate of the receptors

38. An example of receptor-mediated endocytosis: cholesterol uptake • Low density lipoproteins (LDL) is a storage form of cholesterol and the primary vehicle for cholesterol transport in the blood. • Cells need cholesterol for its function and endocytose these LDL particles to bring them into the cells • Cell surface LDL receptor binds to LDL. • The cytosolic portion of the LDL receptor associates with a specific set of clathrin adapters and nucleates the binding of a few clathrin triskelions to form a coated pit. • As more receptors enter the coated pit, more adaptors and clathrin is recruited to form a coated vesicle. • The LDL-containing coated vesicles uncoat and fuse with the early endosome. • The receptor disengages the LDL particle due the pH change and is recycled to the plasma membrane. • The LDL particles are transported to the lysosomes where it is degraded and utilized.

39. Normal and mutant LDL receptors • LDL receptor proteins binding to a coated pit in the plasma membrane of a normal cell. • A mutant cell in which the LDL receptor proteins are abnormal and lack the site in the cytoplasmic domain that enables them to bind to adaptins in the clathrin-coated pits. • Such cells bind LDL but cannot ingest it. Increased risk of a heart attack caused by atherosclerosis.

40. Transport from the trans Golgi netowrk to the cell exterior: Exocytosis • 2 forms of secretory pathways for proteins, lipids, or modified polysaccharides: • Constitutive secretion • This is a continuous pathway for the synthesis and export of proteins, such as those of the ECM (extracellular matrix) or plasma membranes. Operates in all cells. • Regulated secretion • In specialized secretory cells. Vesicles (secretory granules) are prepared and stored for export in response to a signal. e.g. release of hormones, histamine by Mast cells, digestive enzymes & neuro-transmitters at the synapses.

41. Properties of regulated exocytosis: • Proteins are typically very dense (concentrated). • Proteins are selected by signal patches. • Pre-pro-proteins and polyproteins (delayed activation); eg. Preproinsulin activated in secretory granules, trysinogen activated after secretion. • SNARE catalyzed fusion with plasma membrane is inhibited until appropriate signal is received (eg. Hormone, electrical excitation).

42. Formation of secretory vesicles • Secretory proteins become aggregated and highly concentrated in secretory vesicles by two mechanisms: • They aggregate in the ionic environment of the trans Golgi network (often the aggregates become more condensed as secretory vesicles mature and their lumen becomes more acidic. • Excess membrane and lumenal content present in immature secretory vesicles are retrieved in clathrin-coated vesicles as the secretory vesicles mature. • Secretory vesicles bud from the TGN (with clathrin coats), uncoat, move near the plasma membrane, tether, dock, and fuse using PM specific proteins.

43. Polarized cells direct proteins from the trans Golgi network to the appropriate domain of the plasma membrane • Polarized cells: cell with two compositionally distinct and different plasma membranes, the apical and basolateral plasma membrane. • The two different membrane domains are separated by a molecular fence (tight junctions) which prevents proteins and lipids from diffusing between the two domains, so that the compositions of the two domains are different. • The plasma membrane of the nerve cell body and dendrites resembles the basolateral plasma membrane domain of a polarized epithelial cell, whereas the plasma membrane of the axon and its nerve terminals resembles the apical domain of an epithelial cell. • The different membrane domains of both epithelial cell and the nerve cell are separated by a molecular fence, consisting of a meshwork of membrane proteins tightly associated with the underlying actin cytoskeleton (tight junction/axonal hillock).

44. The formation of synaptic vesicles