Mechanism of Vesicular Transport

Transport vesicles play a central role in the traffic of molecules between different membrane enclosedmolecules between different membrane-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 thatselective 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 sorting2 Bi h i l h R tit ti f i l t t i ll f t /2. Biochemical approach: Reconstitution of vesicular transport 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 neuronsneurotransmitters by neurons4. Fluorescent Microscopy

Studies using yeast mutantsStudies 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 areyeast 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.p● 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 pathwayp p y p y

Yeast mutants defective in protein trafficking and sorting

Fig. 17-14

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 ( hi h ll ) b i l h l i i(at which cells cannot grow) but is normal at the lower permissive temperatures, (at which cells grow normally). When transferred from the lower to the higher temperature, these so-called sec mutants accumulate secretor proteins at the point in the path a that is blocked Anal sis ofsecretory proteins at the point in the pathway 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 GolgiRER, 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 analyzedTo 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,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 protein matures it moves sequentially from the cytosol -> y p q y yRER -> ER-to-Golgi transport vesicles -> Golgi cisternae -> secretory vesicles and finally is exocytosed.

Isolation of temperature-sensitive mutants in yeast

Reconstituted vesicular transportThe first cell-free transport system was developed by James Rothman and his colleaguescolleaguesA mutant mammalian cell line that lacked functional N-acetylglucosaminetransferaseat an early stage of N-glycosylation in the at a ea y stage o g ycosy at o t eGolgi. Consequently, the glycoproteins produced by this mutant lacked added N-acetylglucosamine (GlcNAc) units.y g ( )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

i i h i b ifi llproteins it synthesizes can be specifically detected. Transport of these proteins to normal Golgi stacks is signaled by the dditi f di l b ll d Gl NAaddition of radiolabelled GlcNAc.

Cargo selection, Coat proteins, and Vesicle BuddingMost transport vesicles are coated with cytosolic coat proteins, thus called coated vesicle

overview1. Secretory proteins are sorted from proteins targeted for other destinations and

from proteins that need to remain behind2. 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 theymembrane 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 g g p gmembrane

Three types of Coated vesicles

COP-coated vesiclesCOP coated vesicles

COPI : vesicles moving between the Golgi cisternae or retrieval vesicles that returencisternae 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 g pthe cis Golgi network (Retrograde transport)

COPII : carry secretory proteins from the ER y yto 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 t ll l l l f th lextracellular molecules from the plasma

membrane by endocytosis as well as the transport of molecules from the trans Golgi network to endosomes lysosomes or thenetwork to endosomes, lysosomes or the plasma membrane

Different Coat Proteins Act at Specific Points in the Secretory Pathway

removal by retrograde flow maintains identityof ER and Golgiof ER and Golgi

KDEL receptor for soluble proteinsTM proteins with -KKXX interact with COP Itubulation separates membrane and soluble proteinstubulation separates membrane and soluble proteins

Early Secretory Pathway - Forward and Retrograde TrafficEarly Secretory Pathway - Forward and Retrograde Traffic

KDEL-receptors bind toKDEL-receptors bind to KDEL-bearing proteins in the low pH environment of the Golgi and release thatGolgi and release that Cargo in the neutral pH of the ER.

pH probably alters KDELpH probably alters KDEL receptor conformation -regulating cargo binding and inclusion in COPI vesiclesCOPI vesicles.

The formation of clathrin-coated vesicles

Clathrin, GTP-binding proteins (ARF1, ADP-ribosylationfactor1), adaptor proteins1. ARF/GDP on the Golgi membrane2. ARF-GEF (ARF-guanine nucleotide exchange factor) stimulated

the exchange of the GDP for GTP3. ARF/GTP initiates the budding process by recruiting adaptor

i hi h h bi di i f b hproteins, which 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

b d i i i h b dmembrane and initiates the bud5. During the transport, The GTP bound to ARF1 is hydrolyzed to

GDP and the ARF/GDP is released from the membrane for recycling.

6 Th l f ARF1 d h i f i (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

f h f h i l bmost 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

diff t d t t i l l i th blcargeos, different adaptor proteins play a role in the assembly of vesicles for different destinations.

CopI Made of coatamer subunits. Mediates retrieval of proteins p

from Golgi to ER (retrograde transport).

COPI vesicles transport ER resident proteins with KKXX or RRXX psignals.

Uses GTP binding protein ARF (as does clathrin).

CopII Mediates forward movement of vesicles from ER to Golgi (anterograde transport).

R l t d b GTP bi diRegulated by a GTP binding protein Sar1.

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Vesicle fusionTh f i f t t i l ith it t t i l t t f tThe fusion of a transport vesicle with its target involves two types of events1. The transport vesicle must recognize the correct target membrane2. The vesicle and target membranes must fuse, delivering the contents to

the target organellethe 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-transmembrane proteins called SNAREs on the vesicle and target membrane (vSNARE 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 p y p y g yappeared 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 bilayerssufficiently close to destabilize them and result in fusion.

Docking, tethering and fusion to specific target membranes, however, require h ddi i l i b f h b f il f llmuch more additional proteins; members of the Rab family of small GTP-

binding proteins play key roles in this docking. More than 60 different Rab proteins have been identified and shown to function in specific

i l t t (t bl 10 1) Th f ti i t fvesicle transport processes (table 10.1). 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 correctorganelles 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 theThe Rab proteins are carried through the cytosol in their GDP-bound form by GDP-dissociation inhibitor (GDIs). At a membrane, they are removed from GDIs bymembrane, they are removed from GDIs by GDI-displacement factors. Specific guanine-nucleotide 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 Rabpfacmily, so they are responsible for formation of active Rab/GDP complexes at the correct membrane sites. In the absence of the appropriate exchange factor, Rabproteins remain as GDP-bound form and are removed from the membrane by a GDI

d i d h band carried to another membrane

Rab/GTP on the transport vesicle and on the target membrane interacts with effector proteins and SNAREs to assemble a pre-fusion complexWhen the transport vesicle encounters this target membrane the effector proteins linktarget membrane, the effector proteins link the membranes by protein-protein interactions.This tethering of the vesicle to the target

b l b h d l dmembrane 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 todomain 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.h i l h h i i h hiThe simplest hypothesis is that this creates

instability in the lipid bilayers and they fuse.Following membrane fusion, the NSF/SNAP complex disassembles the SNARE complex,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 h f i f h b fthe 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

f fexosytosis, the fusion of a transport vesicle with the plasma membrane, resulting in secretion of the vesicle contents. Many types of exocytosis occur at

ifi t i l ll d t thspecific 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 inyeast, 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 sequentialtheir assembly appears to require sequential interactions among eight exocyst proteins localized on both the transport vesicle and the target membrane sitemembrane site. Interaction of these proteins results in efficient targeting of the transport vesicle to a specific location on the plasma membrane. Several smalllocation 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 play a role in localizing y p y gexocysts ot apical or basolateral membranes or to axons or dendrites

LysosomesyMembrane-enclosed organelles that contain an array of enzymes capable of breaking down all types of biological polymersLysosomes 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 hydrolasesLysosomes 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 b d d d t i l l t ithi th l f ff t dbecause undegraded 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(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 golgitagging 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( 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.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)y )

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 networkIn 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

h d h d i h M i l f id h ll i ksecretory 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,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

6 P t i th t G l i t k d k d i t l th i t dmannose-6-P receptor in the trans Golgi network and packaged into clathrin-coated 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 arelumen 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) pathwayThe mannose 6 phosphate (M6P) pathwaySorting of lumenal proteins can occur by binding transmembrane receptors.

Lysosomal enzymes d f d hmodified with M6P are

bound by the lumenal domain of MP6R.

6 l lMP6R-lysosomal enzyme complexes are recruited into clathrin/AP1 coated pits.

Vesicles deliver the MP6R-lysosomal enzyme complexes to the late

dendosome.

MP6R recycles to the golgi.

Lysosomal enzymes are delivered to lysosomes.