Vesicle formation and targeting is a multi-step process
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1 Vesicle formation and targeting is a multi-step process 2. Formation of coated transport vesicle…3. Targeting and docking to specific compartment…SNAREs and RabsTarget compartmentGTPGDP + Pi(ATP, GTP, and cytoplasmic protein factors…)4. Uncoating…1. Formation of coated buds…GAP and Sar1GEF and Sar1Donor compartmentCoat proteins (“COPs” or “coatomer”)Inhibitors such as GTP-g-S and NEM have been used to map out the steps involved in vesicle transport:Formation of coated buds (requires ATP and cytosol);Formation of coated vesicles (requires ATP and cytosol);Transport and docking to the target membrane;Uncoating (requires GTP hydrolysis; blocked by GTP-g-S); andFusion (blocked by NEM).5. Fusion…SNARE plus other fusion proteins
2 Lecture 16 Vesicle transport and targeting in the secretory pathway COP coated vesiclesSNAREsProtein sorting/targetingSecretion - Golgi to plasma membraneRetention in ERGolgi to lysosomeHow are proteins sorted to appropriate vesicles so that they are transported to proper location?What are the address labels?
3 Two secretory pathways; constitutive and regulated Default pathway for ER/Golgi proteinsIf no address label, then secrete15_28_trans_Golgi_net.jpgECBSignal required to trigger secretory granule fusionExample - neurotransmitter releaseInside lumen is equivalent to outside of cell
4 Regulated secretionSecretory granules containing insulin in pancreatic cellsSignal for release is elevated glucose levels in blood
5 If secretion is default, how are resident ER proteins retained? They aren’t!Ex: BiP is a member of the HSP70 family that functions in the ERBiPKDELKKXXKDEL-RConstituitive secretionSecretory granuleRegulated secretionPlasma membraneERCGNC, M, T GolgiTGNOutsideBiP escapes from ER and must be “retrieved” from the Golgi…C-terminal KDEL in BiP sequence functions as retrieval signal…KDEL-receptors in Golgi direct retrieval/recycling…KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…
6 Summary so far of protein targeting, revisited… Secretion/membrane proteinsProtein targetingCytoplasmRERSignal sequence (hydrophobic a-helix)Vesicle targetingGolgiDefaultKDEL (soluble proteins)KKXX (membrane proteins)Lysosomes?Secretory vesiclesPlasma membraneDefaultA schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.(regulated secretion)(constituitive secretion)See ECB figure 14-5TransportRetrievalHow are proteins targeted to the lysosome?
7 How are proteins sorted to vesicles leaving TGN for lysosome? Lecture 16Vesicle transport and targeting in the secretory pathwayCOP coated vesiclesSNAREsProtein sortingSecretion - Golgi to plasma membraneRetention in ERGolgi to lysosomeHow are proteins sorted to vesicles leaving TGN for lysosome?
8 Lysosomes degrade and recycle macromolecules… ECB 15-34The lysosome of animal cells contains hydrolytic enzymes for degrading/recycling macomolecules. The vacuole of plant cells performs many of the functions of the animal lysosome, as well as regulating turgor pressure.Lysosomes in plant and animal cells contain acid hydrolases (hydrolytic enzymes) for degrading/recycling macromoleculespH of lumen is about 5 - acidic!How are hydrolases and other proteins targeted to lysosomes?
9 I-cell disease helped decipher the signal for targeting proteins to the lysosome Recessive mutation in single gene…Fibroblasts of patients contain large inclusions (I-cells)…Lysosomes lack normal complement of acid hydrolases…All lysosomal enzymes secreted (secretion is the “default” fate for proteins in the ER-Golgi pathway)…Lysosomal enzymes of “wild-type” (normal) cells are modified by phosphorylation of mannose on oligosaccharide (forming mannose-6-phosphate)…Lysosomal proteins of I-cells lack M-6-P…Lysosomal targeting signal resides in carbohydrate!
10 Mannose-6-P targets proteins from Golgi to lysosome Cis GolgiNetwork (CGN)Trans GolgiNetwork (TGN)Addition of M6PTransport via clathrin-coated vesicles to…LysosomeClathrin coatUncoupling(pH 5)MaturehydrolaseRERLysosomal hydrolase (precursor)M6P receptorRemoval of phosphate &proteolytic processing…M6P receptor recycling back to GolgiLysosomal proteins are modified by the addition of M-6-P to their polysaccharides in the cis-Golgi. The trans-Golgi and/or TGN contains M-6-P receptors, whioch bind lysosomal proteins. These receptors are recruited into clathrin-coated pits/vesicles on the TGN, and transported to the endosome, and thence to the lysosome.Addition of M6P to lysosomal enzymes in cis-GolgiM6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated vesiclesPatients with I-cell disease lack phosphotransferase needed for addition of M-6-P to lysosomal proteins in fibroblasts… secreted…
11 Protein targeting, revisited Secretion/membrane proteinsProtein targetingCytoplasmRERGolgiPlasma membraneSignal sequence (hydrophobic a-helix)Vesicle targetingDefault or signal?KDEL (soluble proteins)KKXX (membrane proteins)Secretory vesiclesM6PDefault or signal?A schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.(regulated secretion)(constituitive secretion)LysosomesSee ECB figure 14-5TransportRetrievalNext lecture: endocytosis and clathrin coats
12 Lecture 17 The pathways to the lysosome Phagocytosis Autophagy EndocytosisEndocytosis- The inward limb of membrane cyclingPinocytosisClathrin coated vesiclesReceptor-mediated endocytosis
13 Three pathways to the lysosome PhagocytosisEndocytosisAutophagyECB 15-35
14 Phagocytosis - “cell eating” Performed by specialized “phagocytes:” WBCsPhagocytosis - “cell eating”BacteriumPseudopods1. “Phagocytosis”Entrapment by pseudopodsEngulfment: pseudopods fuse to internalize prey in phagosome…Digestion: phagosome fuses with lysosomePhagosomeVesicles wlysosomal enzymesSome bacteria have evolved to evade digestion in lysosomes, and live as intracellular parasites or pathogens…Myxobacteria tuberculosis (tuberculosis)…Listeria monocytogenes (listeria)…Yersinia pestis (plague)…LysosomeWhere do vesicles with lysosomal contents come from?What is their address label?
15 Autophagy (“Self-eating”); used to recycle worn-out organelles 1. “Phagocytosis”BacteriumPhagosomeVesicles wlysosomal enzymesEndoplasmicreticulumWorn out organelle engulfed by ERLysosomeWorn outmitochondrionAutophagosome2. “Autophagy”
16 Endocytosis: 1. “Phagocytosis” 3. “Endocytosis” 2. “Autophagy” Pinocytosis (“cell drinking”) and “receptor-mediated” endocytosis1. “Phagocytosis”BacteriumPhagosomeEarlyendosomeLateendosome3. “Endocytosis”Vesicles wlysosomal enzymesEndocytotic vesiclesEndoplasmicreticulumLysosomeWorn outmitochondrionAutophagosome2. “Autophagy”Note that vesicles from TGN targeted to lysosome by M6P actually fuse with precursor vesicles/organelles to form lysosome
17 Overview of “pinocytosis” (“bulk” or “fluid-phase” endocytosis) Fluid-phase endocytosis can be followed in live cells with fluorescent dyesProteinaceous coatAs many as ~2500 coated vesicles/min (~2-3% of surface area)!Coated pitGTPGDP+Pi~ 1’: early endosome(pH~6)…ATPADP+PiH+ATPADP+PiATPADP+PiCoatedvesicleUncoating(seconds)…H+~ 5’: late endosome(pH 5.5~6)…Delivery of acid hydrolases from TGN…ATPADP+PiH+~30’: Lysosome(pH<5)…Early endosome - late endosome - lysosome is a continuum
18 EM views - coated pit to coated vesicle Coated pits coated vesiclesECB 15-18
19 Protein coat is “geodesic” clathrin cage Clathrin “heavy chain”“Light chain”3 clathrin “heavy chains”(~ kDa)……plus…3 clathrin light chains(~40 kDa)……form…“Triskelions”…Spontaneously assemble into “geodesic” vesicle coats…15.8-clathrin.mov
20 Components of a clathrin-coated vesicle “cargo”Cargo and receptors we know from COP-coated vesiclesreceptorsclathrinadaptinsmembraneECB 15-19Adaptins - adaptors that bind clathrin and cargo receptor, thereby regulating which cargo gets loaded into clathrin-coated vesicle
21 Pinching off of vesicles requires the protein dynamin ECB 15-19Coated pitbudding“pinching off”(dynamin)uncoatingAssembly of coat causes pit to form due to 3D shape of clathrin coat
22 Dynamin is a GTPase GTP dynamin GDP GTPase that regulates pinching off ECB 15-19GTPGDPdynaminGTPase that regulates pinching offExplains why non-hydrolyzable GTP analogues block endocytosis
23 Clathrin-coated vesicles are rapidly uncoated DynaminAdaptin complexesClathrin“Clathrin-coated pit”By the “clathrin-uncoating ATPase” a member of the HSP70 family of chaperones; requires ATP hydrolysisNaked transport vesicles targeted to endosome…Clathrin and adaptins recycledGTPGDP + PiClathrin uncoating ATPaseATPADP + PiNaked transport vesicleSee ECB figure 15-19To endosome…
25 How do cells take up specific macromolecules? “Receptor-mediated endocytosis”Example: Low-density lipoprotein (LDL), structure in which cholesterol is transported through our bodiesLipid micelle:~800 phospholipids…~500 molecules ofcholesterol…~1500 molecules of cholesterol ester1 copy of apoprotein B…Total mass: ~ 3 x 106 Da
26 Overview of receptor-mediated uptake of LDL ECB 15-3215_32_LDL_enters.jpgLow pH of endosome (~6) causes LDL to dissociate from receptorLDL is transferred to lysosome (fusion of vesicles from TGN)Hydrolytic enzymes cleave LDL, releasing cholesterol to cytoplasm for continued membrane biosynthesis in smooth ERReceptor is recycled back to surface (cycles about every 10 min!)
27 Defects in LDL endocytosis are associated with “familial hypercholesterolemia”… Severe atherosclerosis at early age (strokes and heart attacks in pre-teens)Excess LDL in circulating bloodLDL not properly internalized by cellsRecessive/single gene… encoding plasma membrane receptor for LDL (LDL-receptor or LDL-R)Disease provided insight into mechanism of receptor-mediated endocytosis and identification/function of LDL-receptorMutations in N-terminal domain: LDL-R doesn’t bind LDL…Mutations in C-terminal domain: LDL-R is not internalized…What does this tell you about function of domains of LDL receptor?
28 Domains in LDL receptor Based on MBoC (3) figure 13-53LDLN terminus of LDL receptor binds apoprotein B in LDLC terminus binds adaptinNH2LDL-RPlasma membraneHOOCTyrAsnValProAdaptin complex (four polypeptides)
29 Adaptin complex (four polypeptides) Recruitment of LDL-R to coated pits requires an “endocytosis signal” in cytoplasmic domainBased on MBoC (3) figure 13-53LDLAdaptin complex binds endocytosis signal in cytoplasmic domain of receptor:-NPXY- (Asn-Pro-Val-Tyr) in LDL-RLDL-RPlasma membraneAt least three different adaptin complexes; bind different endocytosis signals on receptorsAdaptins recruit clathrin and initiate coated pit/vesicle formationHOOCTyrAsnEndocytosis signalValProAdaptin complex (four polypeptides)
30 A single coated pit has many different receptors and cargos Low density lipoprotein (LDL)LDL-R1,000s of receptors of many types per coated pit…Same coated pits used for pinocytosis!
31 Summary of “receptor-mediated” endocytosis of LDL Low density lipoprotein (LDL)A single receptor makes hundreds of trips (~10 min/cycle)pH ~7-.7.2LDL-RdynaminGTPGDP+PiEarly endosomeATPADP+PipH ~6pH ~7.2ATPADP+PiH+Uncoating(HSP70 family)CoatedvesicleFusion(Snares)Proton pump in endosome acidifies endosome lumen causing LDL to dissociate from receptorCholesterol ester cleavedCholesterol released for useFree cholesterolLysosome
32 Coats for all reasons: a summary of vesicle coats and functions COPs:Outbound: ER to Golgi transport, intra-Golgi, Golgi to plasma membraneRetrograde: intra-Golgi, Golgi to EREndosomal: early to late/lysosomeClathrin:Plasma membrane to early endosome (endocytosis)Golgi to late endosome/lysosomeDon’t worry about COPI vs II
33 Endosomes sort internalized receptors and ligands ECB 15-33Some ligandsMany receptorsMany ligandsSome receptorsMaternal IgGSecreted IgAOthers15_33_type_of_receptor.jpgTranscytosis - movement of receptor to a different membrane from the one in which it was endocytosed
34 “Transcytosis” moves maternal IgG across epithelia Intestinal lumenMilk ductIgG in milkIgG is “secreted” across the mammary epithelium into milk by transcytosisReceptor-mediated endocytosis from basolateral domain…Secretion from apical membrane domain…Apical membraneIgG receptorTight junctionsApicalEndosomeIgG is transcytosed into the neonate bloodEndocytosis from apical domain and secretion to basolateral membraneBasolateralEndosomeEpithelial cellIgG receptorIgG in bloodPolarized epithelial cells have distinct apical and basolateral endosome compartmentsBasolateral membraneNeonate bloodMaternal blood
35 Protein targeting and trafficking, finale! Secretion/membrane proteinsSignal peptideNucleusNLS: (basic)MitochondriaProtein targetingECB considers these all to be signal sequencesNES: (L-rich)CytoplasmChloroplastsPeroxisomesRERGolgiPlasma membraneSignal sequenceTransit peptideSKL at C term.Vesicle targetingKDEL (soluble proteins)KKXX (membrane proteins)EndosomesM6PSecretory vesiclesDefaultA schematic summary of protein targeting and vesicle trafficking in a typical eukaryotic cell.(regulated secretion)(constituitive secretion)EndocytosissignalLysosomesTransportRetrievalEndocytosis: From plasma membrane to endosome to lysosome…
36 Membrane flow during exocytosis and endocytosis is a delicate balance Golgi apparatusEndosomeERLysosomeOriginal surfaceEndocytosis internalizes membrane ~2-3% per minute…Entire membrane is recycled in less than 1 hr…Block endocytosis, exocytosis continues:Block exocytosis, endocytosis continues:membrane area grows…membrane area shrinks…
37 Intermediate filaments: Next lecture…“Cytoskeleton”Intermediate filaments:Cell structureMicrofilaments: MuscleOrganelle transport in plantsMicrotubules:Cilia and flagellaOrganelle transport in animalsECB 1-20