Mechanism of Vesicular Transport Transport vesicles play a central role in the traffic of molecules between different membrane-enclosed enclosed compartments. The selectivity of such transport is therefore a key to maintaining the functional organization of the cell. The specificity of transport of transport is based on the selective packaging of the intended cargo into vesicles that recognize and fuse only with the appropriate target membrane.
Experimental Approaches to understanding vesicular transport 1. Isolation of yeast mutants that are defective in protein transport and sorting 2. Biochemical i approach: Reconstitution of vesicular transport t in cell-free system / isolation of enzymes and proteins involved in protein processing and sorting 3. Studies of synaptic vesicles, which are responsible for the regulated secretion of neurotransmitters by neurons 4. Fluorescent Microscopy Studies using yeast mutants Advantageous because they are readily amenable to genetic analysis Randy Schekman and his colleagues have pioneered the isolation of yeast mutants defective in vesicular transport including mutants that are defective at various stages of protein secretion (sec mutants), mutants that are unable to transport proteins to the vacuole, and mutants that are unable to retain resident ER proteins. The isolation of such mutants led directly to the molecular cloning and analysis of the corresponding genes, thereby identifying a number of proteins involved in various steps of the secretory pathway
Fig. 17-14 Yeast mutants defective in protein trafficking and sorting
Other studies have been done using yeast mutants which has even defined the pathway by which secretory proteins mature. A large number of temperature sensitive mutant yeast strains were identified in which the secretion of all proteins is blocked at the higher, nonpermissive temperature (at which h cells cannot grow) but is normal at the lower permissive i temperatures, (at which cells grow normally). When transferred from the lower to the higher temperature, these so-called sec mutants accumulate secretory proteins at the point in the pathway a that is blocked. Analysis of such mutants identified five classes (A-E), corresponding to the five steps in the secretory pathway, in which secretory proteins accumulate in the cytosol, RER, small vesicles taking proteins from the ER to the Golgi complex, Golgi cisternae, or secretory vesicles. To determine the order of the steps in the pathway, researchers analyzed double sec mutants. For instance, when yeast cells contain mutations in both class B and class D functions, proteins accumulate in the RER, not in the Golgi cisternae. Since proteins accumulate at the earliest blocked step, this finding shows that class B mutations must act at an earlier point in the maturation pathway than class D mutations do, These studies confirmed that as a secretory yprotein matures it moves sequentially from the cytosol -> RER -> ER-to-Golgi transport vesicles -> Golgi cisternae -> secretory vesicles and finally is exocytosed.
Isolation of temperature-sensitive mutants in yeast
Reconstituted vesicular transport The first cell-free transport system was developed by James Rothman and his colleagues A mutant mammalian cell line that lacked functional N-acetylglucosaminetransferase at an early stage of N-glycosylation o in the Golgi. Consequently, the glycoproteins produced by this mutant lacked added N- acetylglucosamine yg (GlcNAc) units. Golgi stacks isolated from a virus-infected mutant cell line unable to catalyze the addition of GlcNAc to N-linked oligosaccharides are mixed with Golgi stackes from a normal cell line. Because the mutant cells were infected by a virus, the proteins it synthesizes can be specifically detected. Transport of these proteins to normal Golgi stacks is signaled by the addition of radiolabelled ll d GlcNAc.
Cargo selection, Coat proteins, and Vesicle Budding Most transport vesicles are coated with cytosolic coat proteins, thus called coated vesicle overview 1. Secretory proteins are sorted from proteins targeted for other destinations and from proteins that need to remain behind 2. The caots assemble as the secretory protein-containing vesicles bud off the donor membrane and are generally removed from the vesicle in the cytosol before they reach their target 3. At the target membrane, the vesicles dock and fuse with the membrane, emptying their lumenal cargo and inserting their membrane proteins into the target membrane
Three types of Coated vesicles COP-coated vesicles COPI : vesicles moving between the Golgi cisternae or retrieval vesicles that returen resident ER proteins marked by the KDEL or KKXX retrieval signals back to the ER from the ER-Golgi interamediate compartment or the cis Golgi network (Retrograde transport) COPII : carry secretory proteins from the ER to the ER-Golgi intermediate compartment or Golgi apparatus, budding from the transitional ER and carrying their cargo forward along the secretory pathway Clathrin-coated vesicles: the uptake of extracellular l molecules l from the plasma membrane by endocytosis as well as the transport of molecules from the trans Golgi network to endosomes, lysosomes or the plasma membrane
Different Coat Proteins Act at Specific Points in the Secretory Pathway
removal by retrograde flow maintains identity of ER and Golgi KDEL receptor for soluble proteins TM proteins with -KKXX interact with COP I tubulation separates membrane and soluble proteins
Early Secretory Pathway - Forward and Retrograde Traffic 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.
The formation of clathrin-coated vesicles Clathrin, GTP-binding proteins (ARF1, ADP-ribosylation factor1), adaptor proteins 1. ARF/GDP on the Golgi membrane 2. ARF-GEF (ARF-guanine nucleotide exchange factor) stimulated the exchange of the GDP for GTP 3. ARF/GTP initiates the budding process by recruiting adaptor proteins, which h then serve as binding sites for both transmembrane receptors and for clathrin. 4. Clathrin actually plays a structural role in vesicular budding by assembling into a basketlike lattice structure that distorts the membrane and initiates i i the bud 5. During the transport, The GTP bound to ARF1 is hydrolyzed to GDP and the ARF/GDP is released from the membrane for recycling. 6. The loss of ARF1 and the action of uncoating enzymes (e.g. Hsc70) weakens the coopertive binding of the clathrin coat complex such as by elicit conformational change of clathrin, allowing chaperone proteins in the cytoplasm to dissociate most of the coat from the vesicle membrane * clathrin-coated vesicles exit the trans Golgi for different destinations: endosomes, lysosomes, or different plasma membrane domains. Since these targets require specific cargeos, different adaptor proteins play a role in the assembly of vesicles for different destinations.
CopI Made of coatamer subunits. Mediates retrieval of proteins from Golgi to ER (retrograde transport). COPI vesicles transport ER resident proteins with KKXX or RRXX signals. Uses GTP binding protein ARF (as does clathrin). CopII Mediates forward movement of vesicles from ER to Golgi (anterograde transport). Regulated by a GTP binding protein Sar1. 14
Vesicle fusion The fusion of a transport t vesicle with its target t involves two types of events 1. The transport vesicle must recognize the correct target membrane 2. The vesicle and target membranes must fuse, delivering the contents to the target organelle. SNARE hypothesis by Rothman Vesicle fusion is mediated by interactions between specific pairs of transmembrane proteins called SNAREs on the vesicle and target membrane (v- SNARE and t-snare respectively) This hypothesis was supported by the identification of SNAREs that were present on synaptic vesicles and by the finding of yeast sec mutants that appeared to encode SNAREs required for a variety of vesicle transport events. Basically, SNAREs are required for vesicle fusion with a target membrane and that SNARE-SNARE pairing provides the energy to bring the two bilayers sufficiently close to destabilize them and result in fusion. Docking, tethering and fusion to specific target membranes, however, require much more additional i proteins; members of the Rab family of small GTPbinding proteins play key roles in this docking. More than 60 different Rab proteins have been identified and shown to function in specific vesicle transport t processes (table 10.1). 1) They function in many steps of vesicle trafficking, including interating with SNAREsto regulate and facilitate the foramtion of SNARE/SNARE complex
Individual Rab or combinations of Rab proteins mark different organelles and transport vesicles, so their localization on the correct membrane is key to establishing the specificity of vesicular transport The Rab proteins are carried through the cytosol in their GDP-bound form by GDPdissociation inhibitor (GDIs). At a membrane, they are removed from GDIs by GDI-displacement factors. Specific guaninenucleotide exchange factors then convert Rab/GDP to the active Rab/GTP state. Individual guanine nucleotide exchange factors are localized to specific membranes and act on specific members of the Rab facmily, so they are responsible for formation of active Rab/GDP complexes at the correct membrane sites. In the absence of the appropriate exchange factor, Rab proteins remain as GDP-bound form and are removed from the membrane by a GDI and carried to another membrane
Rab/GTP on the transport vesicle and on the target membrane interacts with effector proteins and SNAREs to assemble a prefusion complex When the transport vesicle encounters this target membrane, the effector proteins link the membranes by protein-protein interactions. This tethering of the vesicle to the target membrane stimulates Rab/GTP hydrolysis and allows the contact between v- & t-snares. All SNAREs have along central coil-coil domain and this domain binds strongly to other coil-coil domains and, in effect, zips the SNAREs together, brining the two membrane into nearly direct contact. The simplest hypothesis h is that this creates instability in the lipid bilayers and they fuse. Following membrane fusion, the NSF/SNAP complex disassembles the SNARE complex, allowing the SNAREs to be reused for subsequent rounds of vesicle transport. As the energy of SNARE-SNARE interaction drives the fusion of the membrane, energy from hydrolysis of ATP is required to separate the SNAREs.
Specific types of jusion may involve specialized sites on the plasma membrane. One of these is exosytosis, the fusion of a transport vesicle with the plasma membrane, resulting in secretion of the vesicle contents. Many types of exocytosis occur at specific protein complexes, called exocysts, on the plasma membrane. This eight protien complex was first discovered to be required for secretion in the yeast, but is also plays an important role in secretion in polarized mammalian cells. The structure of exocysts is not well understood but their assembly appears to require sequential interactions among eight exocyst proteins localized on both the transport vesicle and the target membrane site. Interaction of these proteins results in efficient targeting of the transport vesicle to a specific location on the plasma membrane. Several small GTP-binding proteins are also associated with exocysts and these are involved in vesicle docking and fusion but others may yplay a role in localizing exocysts ot apical or basolateral membranes or to axons or dendrites
Lysosomes Membrane-enclosed organelles that contain an array of enzymes capable of breaking down all types of biological polymers Lysosomes function as the digestive system of the cell, serving both to degrade material taken up from outside the cell and to digest obsolete components of the cell itself. In their simplest form lysosomes are visualized as dense spherical vacuoles and display considerable variation in size and shape as a result of differences in the materials that have benn taken up for digestion Lysosomal acid hydrolases Lysosomes contain about 50 different degradative enzymes that can hydrolyze proteins, DNA, RNA, polysaccharides, and lipids. Mutations in the genesthat endoce these enzymes are responsible for more than 30 different hyman genetic diseases, which are called lysosomal storage diseases because undegraded d d material accumulates within thelysosomes of affected individuals. Most of theses diseases result from deficiencies in single lysosomal enzyme (Gaucher disease results from a mutation in the gene that encodes a lysosomal enzyme required for the breakdown of glycolipids. I cell disease is caused by a deficiency in the enzyme that catalyzes the first step in the tagging of lysosomal enzymes with mannos-6-phosphate in the golgi apparatus. The result is a general failure of lysosomal enzymes to be incorporated into lysosomes
Most lysosomal enzymes are acid hydrolases, which are active at the acidic ph (~5) that is maintained within lysosomes but not at the neutral ph characteristic of the rest of the cytoplasm: protection against uncontrolled digestion of the contents of the cytosol even if lysosomal membrane were broken down. To maintain their acidic internal ph, lysosomes concentrate H+ ions using a proton pump in the lysosomal membrane (a hundred fold higher H+ inside the lysosome)
Endocytosis and lysosome formation One of the major functions of lysosomes is the digestion of material taken up form ouside the cell by endocytosis (chapter 13). In particular, lysosomes are formed when transport vesicles from the trans Golgi network fuse with endosomes, which contain molecules taken up by endocytosis at the plasma membrane. The formation of endosomes and lysosomes thus represents an intersection between the secretory pathway and the endocytic pathway. Materials from outside the cell is taken up in clathrin-coated endocytic vesicles, which bud from the plasma membrane and then fuse with early endosomes. Membrane components are then recycled to the plasma membrane (chap 13) and the early endosomes gradually mature into late endosomes, which are the precursors to lysosomes. One of the important changes during endosome maturation is the lowering of the internal ph Acid hydrolases targeted to lysosomes by mannose-6-phosphate are recognized by mannose-6-p receptor in the trans Golgi network and packaged into clathrin-coated t vesicles. Following fusion of the vesicles with late endosomes, the acidic ph causes the hydrolases to dissociate from the receptor. The hydrolases are thus released into the lumen of the endosome, while the receptores remain in the membrane and are eventually recycled to the Golgi. Late endosomes then mature into lysosomes as they acquire a full complement of acid hydrolases
The mannose 6-phosphate (M6P) pathway Sorting of lumenal proteins can occur by binding transmembrane receptors. Lysosomal enzymes modified df dwith M6P are bound by the lumenal domain of MP6R. MP6R-lysosomal l enzyme complexes are recruited into clathrin/ap1 coated pits. Vesicles deliver the MP6R-lysosomal enzyme complexes to the late endosome. MP6R recycles to the golgi. Lysosomal enzymes are delivered to lysosomes.