Membrane trafficking Flashcards
(24 cards)
Secretory pathway, mapping and the ER
Organelles communicate via tubular/vesicular transport intermediates- bud from donor compartment. 2 main trafficking pathways- secretory (AKA biosynthetic- new proteins ER->Golgi-> plasma mem) and endocytic (endocytosis, series/ endosomal compartments, lysosome/ vacuole (fungi+ plants). Connected via trans-Golgi network+ endosomes, many routes bidirectional. Anterograde(to mem) balanced w/ retrograde-> homeostasis.
Mapping secretory pathway- 1st by Palade’s pulse-chase exps: pancreatic cells incubated w/ radio aas for few mins (pulse), then incubated w/unlabelled medium for diff times (chase)-> autoradiography+ EM show in few mins labelled proteins in ER, then Golgi, then secretory granules. Now, use fluor proteins/lipids for live tracking.
ER: sheet-like part studded w/ ribosomes (rough)+ tubular smooth. Main site/ lipid biosynth, provides env for nascent protein folding- packed w/ chaperones, e.g. binding protein BiP- help folding, prevent aggregation. Oxidising compartment- cysteine sulfhydryls form cov S-S bonds, helped by protein disulfide isomerases (PDI). Many proteins glycosylated (invariant glycan-> Asps in consensus NXS/T, X= any but Pro)- helps folding+ chaperone binding. Quality ctrl- only correctly folded proteins-> Golgi, others degraded in cytosol. Export in ER exit sites in smooth ER, cargo packaged in COPII vesicles.
ERGIC, Golgi
ERGIC (ER Golgi intermediate compartment): COPII vesicles from ER fuse (homotypic fusion)-> vesicular tubular clusters (VTCs)-> further fusion- these clusters= ERGIC- 1st comp for protein recycling for return to ER, incl ‘escaped’ proteins+ trafficking machinery to be recycled- retrograde transport by COPI vesicles. ERGIC attaches MTs via dynein-> Golgi, where either fuses w/ cis cisterna (area sometimes called cis-Golgi network) or have further homotypic fusion-> new cis cisterna.
Golgi- stack/ flat, fenestrated mem sacks (cisternae) of variable # (~3-20). Stack has cis, medial+ trans compartments of diff f(x)+ partly distinct protein+ lipid complements. Secretory proteins move cis->trans, get post-translational mods- trimming, addition/extension/glycans, sugar phosphorylation, sometimes proteolytic cleavage. diff mods in each Golgi part, can be used to monitor protein progress. Also synth/ most extracellular polysaccharides-glycosaminoglycans+ cell wall (plant+ fungi) polysaccharides here.
Protein mods, golgi transport models, TGN
Mods: cis: -Man, Pi. Medial: -Man, +GlcNAc. Trans: sulphation, +Gal, +Sialic acid.
Models for golgi transport: vesicular transport model: secretory cargo transported by vesicles, ERGIC fuses w/ cis- supported by e- micrographs show vesicles budding from cisternae edges, in vitro assays. Cisternal maturation model: cisternae exchange protein complements over time, cargo stays in same cisterna (enzyme transport trans->cis), ERGICs form new cis- supported by yeast live cell (where cisternae don’t stack) imaging showing maturation- additional vesicular/tubular antergograde transport may still occur. No consensus, possibly both partly right.
Trans Golgi network (TGN): in cisternal maturation model, this is trans-cisterna in process of conversion to secretory carriers (final maturation step)- has characteristics of stable compartment. Some organisms, f(x) as endosome. Major sorting station, protein paths diverge here. Most proteins direct-> mem (bulk/default secretion, may req no more sorting signals). Live imaging shows secretion mediated by tubular carriers (not known what (if any) protein coat these req) ‘pulled’ out/ TGN by molecular motors. Polarised cells have 2 mem domains (apical/basolateral, axonal/dendritic) w/ diff compositions. Many cells- sporting in TGN into diff carriers e.g. vesicular stomatitis virus envelope glycoprotein (VSV-G) sorted to basolaterally- destined vesicles. Some proteins conc in secretory vesicles (/granules)- only fuse mem as signal response, e.g. insulin/ ACh. Some proteins-> clathrin-coated vesicles/ other types/ non-clathrin coated vesicles, delivered to endosomes. TGN-endosome route= interface between secretory+ endocytic pathways.
ER targeting
ER targeting: Blobel+ Dobberstein’s in vitro targeting system- secreted Ig light chains made as larger polpypeps when synth in vitro (seen on SDS-PAGE) than in vivo- if add ER fractions in vitro, get shorter in vivo version. Translation w/ ER mems-> proteins protected from proteases by mems (unlike w/out ER)-> signal hypothesis for ER targeting. Most proteins for ER targeting have N-term secretion signal seq in 1st part/protein to leave ribosome- recognised by signal recognition particle SRP, binds+ temporarily halts translation. SRP binds SRP receptor in ER mem, nascent polypep transferred to Sec61 translocon, translation restarts, protein co-translationally transferred across ER mem. Secretory proteins’ signal pep cleaved off (signal peptidase).
ER sorting: signal seq and SRP
Signal seq+ SRP: yeast secretes invertase, allowing growth on sucrose. If N-term signal/ invertase replaced w/ random seqs, ~20% yeast survive (secrete some), showing almost any N-term hydrophobic seq sufficient to (inefficiently?) target invertase to ER. Most signal seqs also have protease cleavage site- consensus AXA. Ala often replaced by other small aa (G, S). SRP+SRP receptor have GTP binding domains. SRP=multi-protein complex w/ structural 7S RNA. Cross-linking exps show 54kD subunit binds signal. Crystal structure shows hydrophobic groove- can accommodate various hydrophobic seqs. SRP actions reg by reg binding+ hydrolysis/ GTP- premature hydrolysis releases incorrectly targeted proteins before transferred to translocon. Weak signal-SRP interaction-> GTP easily hydrolysed, polypep displaced. Strong interaction- polypep transferred ->translocon, enter ER, then GTP hydrolysed (dissociate SRP from receptor)
Translocation and topologies
Translocation: translocon=trimer of Sec61alpha, beta+gamma. Crystal/ cryoEMs show it forms constricted porein ER mem. When inactive, central opening ‘plugged’ by sec61a helix- prevent ER/cytosolic ion gradient equilibration. When rib w/ appropriate nascent chain docked @rib binding site, plug moved from pore ring. Topologies (orientations+# transmem-domains) of insertion vary, determined in biosynth: soluble proteins enter ER completely. Single pass TMD: type 1 has NTD in ER lumen, type II has CTD in ER lumen. Multiple TMDs- protein comes in+ out of ER lumen/passes mem 2+ times. If protein soluble+ secreted, has cleavable signal peptide. Some single TMD proteins have uncleavable signal anchors- SEC61 opens laterally, allows hydrophobic signal diffusion into ER mem; anchor becomes TMD. Recently discovered ER mem protein complexes (EMC, BOS, GEL, PAT) assist integration/some proteins, esp w/2+ TMDs+ w/ short translocated regions flaking 1st TMD, incl additional insertase activities so some TMD not inserted via SEC61.
Protein sorting and yeast sec mutants
Protein sorting: fundamental principles for compartment composition maintenance+ exchange between compartments: sorting req transport intermediates (vesicles, tubules)+ machinery to make them (e.g. proteins inducing mem curve+ protein coats); protein sorting into transport intermediates selective- necessitates signals+ recognition machinery; sorting imperfect-need 2+ sorting stages of diff composition; intermediated fuse only w/ specific target organelle.
Yeast sec mutants: Schekman+ Novick- genetic approach to ID secretory machinery. Sec deficient yeast become denser. ID’d 23 necessary genes SEC1-23. Ts mutants blocked various secretion stages- mutant morphology (under EM), glycosylation defects, epistasy-> order steps in pathway. More sec genes since found.
ER export: yeast in vitro ER budding system allowed probing of mech/export- found ER derived vesicles rich in secretory cargo (vs ER-resident proteins). In vitro budding req cytosol, suggesting proteins outside ER necessary to form vesicles- incl Sec13+ 31, 23+ 24.
COPII vesicles
COPII vesicles: Sec23/24 dimer=cargo selective adaptor, inner coat. Sec 13/31 dimer- structural function, outer coat. In vitro, sufficient to form empty COPII cages. In vivo COPII recruitment to ER mem: GD-TP exchange in small GTPase Sar1- switch from cytosolic to ER mem-interacting form. Sar1-GTP recruits Sec23/24 dimers, which recognise cytosolic domains/ integral mem proteins for secretion (contain Di-acidic seq motifs (DXD, DXE, EXE)), recruit Sec13/31. Di-acidic seqs mediate incorporation into COPIIs, but sorting signals for ER exit generally poorly understood. ER prevents direct luminal cargo/COPII coat interaction, so soluble proteins req transport receptors like p24- integral me proteins w/ cytosolic domain recognised by COPII coat; luminal domain binds soluble cargo, dragging cargo into budding COPIIs. Cargo-receptor interaction sorting signals poorly understood- glycosylation/cargo may have role. Mem deformation+ vesicle pinching. Sar1 has intrinsic GTPase activity, served as timer for uncoating. GTP hydrolysed-> Sar-GDP can’t bind mem-> COPII coat disassembles, cargo delivered to ERGIC.
Escaped ER protein retrieval and COPI vesicles
Escaped ER protein retrieval: in ERGIC, cargo receptors for ER export recycled back via retrograde pathway- also for mem homeostasis. ER proteins ‘leak’ out to ERGIC, returned by COPI.
COPI vesicles: Munro+ Pelham saw many soluble ER proteins have 4 aa seq KDEL/HDEL@ C-term. Deletion-> secretion. When fused to invertase, it’s retained- hence K/HDEL necessary+ sufficient for ER retention. Also in C-term/ some Type II ER transmem proteins (but these more commonly have dibasic aa cytosolic motifs, incl RR (Type II), KKXX (Type I)-also necessary+ sufficient for ER retention). Lumenal K/HDELs can’t be directly sorted by cytocolic coat, req transmem receptor- ID’d by yeast screen. Yeast cells expressing invertase-HDEL can’t grow on sucrose (enzyme retained); HDEL receptor mutations rescue this-> Erd2p receptor.
Cytosolic retention motifs, erd2 receptor cycles
Cytosolic retention motifs can directly interact COPI coat-> incorporation. Cosson+ Letourneur showed mutation of coatomer subunits-> secretion/ER proteins w/ KKXX motifs. Core COPI coat machinery: heteroheptameric complex (coatomer)+ small GTPase Arf1 (similar role in recruitment to Sar1, can be inhibited by Brefeldin A). Coatomer recognises cargo, has structural f(x)s. Budding process analogous to COPII.
Erd2 receptor cycles between ER, ERGIC, Golgi. Recent structures suggest: lower luminal pH in Golgi increases Erd2 cargo affinity; receptor packaged into COPI by exposing basic patch on cytosolic face+ bound K/HDEL protein retrieved to ER. ER higher pH-> lower affinity-> cargo release-> conf change, COPII diacidic motif revealed-> forward traffic. COPI retrograde traffic both from cis-Golgi+ ERGIC-> ER- 2nd round/ failsafe for sorting (needed due to low fidelity of process)
NB: plants+ fungi don’t have ERGIC, traffic ER-> Golgi+ Erd2 recycling Golgi-> ER only.
sorting in the golgi stack and TGN
Sorting in Golgi stack: sorting signals allow each golgi protein to find its cisterna- poorly understood, some models proposed,
1) Many golgi proteins-> large multiprotein complexes. Kin-recognition for sorting+ retention, esp if complexes too large to enter anterograde transport vesicles (contradicts cisternal maturation model which req vesicular transport/Golgi enzymes)
2) Sorting proteins by lipid environment. Sterol+ sphingolipid content/Golgi mems increases cis to trans, affecting mem properties. Properties (aas, seq+length) of TMDs/ Golgi enzymes allows partitioning into preferred cisterna, excluded from unfavourable envs. Selective retrograde COPI transport has import role in Golgi sorting, involves Vps74p/GOLPH3 interaction w/ cytosolic tails, Golgi PI-4-P lipids+ COPI
Sorting in TGN: Sorting to reg secretory vesicles likely involves kin recognition+ retrograde sorting/unwanted proteins from immature secretory vesicles. Targeting to plasma mem (default secretion) doesn’t always req extra signals, but in polarised cells cytoplasmic signals can direct to basolateral secretory pathway; sorting into apical sec vesicles may involve affinity for specialised lipid domains in TGN (lipid ‘rafts’). Targeting to endosome by clathrin-coated vesicles, req specific sorting signals recognised by adaptor proteins.
Endocytosis mechanism and mapping
Mechanism: mem invagination, intracellular vesicle formation. Most endocytosed material quickly regurgitated, balance w/ exocytosis to maintain cell vol+ recycle mem lipids. Some internalised material-> lysosome, digested. Several types of endocytosis: clathrin-coated pit- mediated best understood. Also phagocytosis (large particles), micropinocytosis (large amount extracellular fluid by me m ruffling), caveolae (flask-shaped invaginations, can bud off+ internalise cargo), probably several other types.
Mapping the pathways: pulse-chase exps- typical tracers incl gold-labelled proteins (visualise w/ EM). In mins, internalised tracers labeltubular compartments near plasma mem (early endosomes/EE), then in ~1hr late endosomes (LE)- less tubular than EE, often have small intralumenal vesicles therefore sometimes called multivesicular bodies (MVBs). Finally, ~4hr-> lysosomes labelled, give dense appearance w/ EM- sometimes 2+ internal mem layers (whorls) discernible.
EE maturation and LE-lysosome fusion
EE maturation: 2 successive pulses label diff Ees, labels only mix in LE. Suggests EE not static, ‘matures’ to LE state. EE->LE converstion seen w/ live imaging- protein marker of EE (Rab5) replaced w/ LE marker Rab7. Maturation in 5 stages: 1) EE receives cargo (many incoming transmem proteins (e.g. cell surface receptors) recycled back to mem. EE also gets input from TGN to connect secretory+ endocytic pathways. 2) EE stops aaccepting cargo, TGN input+ recycling continue-> EE proteins replaced w/ LE ones. 3) proton-pumping v-ATPases acidify endosome. 4) small vesicles bud into endosome. Outward budding recycles proteins to TGN. 5) LEs undergo homotypic fusion, exchanging contents.
LE-lysosome fusion: lysosomes have variety/hydrolytic enzymes, can break down most bio macromolecules. Hydrolases f(x) only @ low pH, maintained @4.5-5. Digest material+ signal nutrient status to cell, functioning as cellular recycling station. Some specialised ones secrete contents @ cell mem. LE-> lysosome transport by direct fusion- different types observed- range from transient ‘kiss-and-run’ to forming hybrid organelle ‘endolysosome’, in which LE+lysosome contents mix, LE contents degraded. Lysosome reformed by maturation, involving vesicular recycling.
Clathrin-mediated endocytosis
Clathrin-mediated endocytosis: extracellular fluid phase endocytosed randomly, but cell surf proteins taken up v selectively by mech of clathrin-mediated endocytosis (CME). Clathrin-coated vesicles= cargo-selective adaptor protein complex 2 (AP-2)+ structural protein clathrin arr. In trimers (triskelia) assembled into baskets/hexagons+ pentagons like football. Cell surf receptor cytoplasmic tails have sorting signals recognised by AP-2 (or alt adaptor that binds AP-2). AP-2 recruits free triskelia, assemble lattices on mem that invaginate-> clathrin-coated pits (CCPs). Proteins for endocytosis conc in CCPs. Thin stalk forms, connects almost-complete vesicle w/ cell mem. GTPase dynamin forms collar around stalk, pinch vesicle off. Auxilin+ chaperone hsc70 use ATP to mediate clathrin coat shedding, the AP-2 can diffuse back to cytosol, leaves fusion-competent vesicle to fuse EE.
Constitutive receptor mediated uptake
Constitutive receptor mediated uptake, recycling: cell surf receptors mediate nutrient internalisation, incl transferrin (Tf) receptor for iron uptake, low-density-lipoprotein (LDL) for cholesterol- both w/ short linear sorting signals in cytoplasmic tails. Tf receptor has canonical+ v common AP-2 binding motif YXXphi (x=any, phi=bulky hydrophobic aa). LDL rec has NPXY motif recognised by alt adaptor Dab2 that binds AP-2, drags LDL rec into CCPs. Once internalised, LDL-> EE- mildly acidic (pH6), reduces LDL rec affinity for cargo, free LDL released into EE lumen while empty rec recycled to mem likely by ‘geometry-based sorting’ into mem tubules of EE- may also go via recycling endosome (slower). LDL-> LE-> lysosome, degraded. Tf rec pathway analogous. Both LDL+ Tf rec internalised+ recycled regardless of whether cargo bound (constitutive endocytosis)
ligand-induced uptake and degradation
Ligand-induced uptake+ degradation: epidermal growth factor (EGF) e.g.- EGF binding causes EGFR ubiquitination, recruiting AP-2 and conf change in EGFR monomers-> dimerize, bringing 2EGFRs together-> auto-Pi on Tyr-> PiTyr=docking site for other proteins incl CbI (E3 UQ ligase that adds UQ to Lys in activated EGFR tail). UQ recognized by alt adaptor Epsin, binds AP-2+ drags EGF into CCPs. When EGFR in EE, UQ=sorting signal recognised by ESCRT- its complexes 0, I+ II detain EGFR, preventing recycling, then coral receptors in small patch of mem. ESCRT III polymerisation-> inward budding/corralled mem patch-> intralumenal vesicle, terminating signalling activity/ EGFR- protein accessible for digestion once in lysosome (conveyor belt model/MVB formation) (some ESCRTs used for other mem budding/scission events, e.g. cytokinesis). Inward budding of intralumenal vesicles mech diff from clathrin coated vesicle formation- both req cytosolic factors, but ESCRT-mediated budding produces bare/uncoated vesicle (hence ESCRT proteins avoid degradation by lysosome)
TGN to endosome transport
TGN to endosome transport: vesicles bud off TGN, deliver cargo to EE, follow lysosomal pathway/recycling route to cell mem- req sorting in TGN to divert EE cargo from bulk of default secreted material, e.g for lysosomal hydrolases- soluble proteins w/ cleavable signal peptide that directs co-translational import to ER, where they’re N-glycosylated. Lysosomal hydrolases have signal patch (signal from 3D fold of protein) recognised in Golgi by N-glycan modifying enzymes-> glycans Pi’d, display terminal mannose-6-Pi (M6P).
In TGN, M6P recognised by its receptor- an integral mem protein w/ 2+ sorting motifs in cytoplasmic tail, incl signals for binding clathrin adaptors. Clathrin-coated vesicles can also bud from TGN using same pool/ clathrin but diff cargo adaptors- GGAs (recognise DXXLL motifs)+ AP-1 (recognises YXXphi like AP-2),which can sort M6P receptor. TGN -derived clathrin-coated vesicle formation req small GTPases (like Arf1).
Vesicles carrying loaded M6P-receptor fuse w/ EEs- as it matures, pH drops, favours dissociation/hydrolase from M6PR. M6PR directed by WLM-motif, becomes conc in retrograde transport carriers leaving LE- carriers use retromer protein complex as coat, return empty M6PR to TGN. Hydrolase-> lysosome.
M6P receptor can also take alt route via cell mem, then internalised via AP-2/CCPs- route can retrieve lysosomal enzymes mis-sorted @ TGN+ accidentally secreted.
M6PR has 2+ sorting signals, allowing cycling between TGN/cell mem/endosomes. Many post-Golgi integral mem proteins cycle similarly w/ diff steady state distributions. Some organisms, incl plants, appear to have TGN+ EE f(x) in same organelle.
Sorting summary table
Compartment ID and membrane fusion
Factors determining coat protein recruitment: diff protein composition/diff compartment mems; distinct lipid compositions attracting diff sets/interacting proteins; vesicle formation coupled to cargo+ trafficking machinery presence, preventing empty vesicles forming. E.g. Sar1 for COPII formation needs to be activated w/ GD-TP exchange mediated by GEF Sec12- latter=mem protein of ER, so Sar1-GDP only changed to GTP near ER mem, which it then binds. Sar1-GTP signal to bind COPII to ER mem but interaction not strong- stabilised by 2nd interaction by cytoplasmic tails/cargo molecules- bind COPII coat (Sec23/24)- acts as coincidence detector: only sufficient quantities of Sar1-GTP+ cargo allow COPII assembly+ budding.
Phosphoinositides (PIPs)= lipid class specifying compartment ID. Pi/inositol ring in 3/4/5 position-> 7 species. Pi -vely charged-> PIPs have diff surface charge profiles+ direct v specific protein interactions. Localise to diff compartments (see diagram). PIPs easily interconverted by (de)pi, so mems can change ID markers, e.g. during vesicle transport.
AP-2 samples plasma mem- has 2 binding sites recognising PI(4,5)P2, allow it to associate transiently w/ plasma mem. Interaction stabilised by cargo-> vesicle formation. AP-2= coincidence detector to reset system after budding; Pi4,5P2 de-pi’d, lowering AP-2 affinity for mem, aiding uncoating.
Vesicle fusion specificity mechanisms
Vesicle fusion specificity ctrl @ 3 levels:
Vesicle transport: long-distance transport by motors+ cytoskeleton. MTs+ F actin polar, determine transport direction. Cytoskeleton rear. Can direct transport- e.g. polarisation of cytotoxic T cell- MTOC relocated to direct all secretory traffic to contact site
Tethering factors (pathway specific)+ Rab GTPases: tethering factors= proteins binding vesicle+ target mem simul.- 2 classes: long rod-like coiled-coil proteins (EEA1, p115, Golgins) capture vesicles + make collision w/ target mem more likely; multiprotein complex tethers (TRAPP, COG, HOPS, Exocyst) bring vesicle_ target mem together- some directly interact fusion machinery. Tether specifically recognises vesicle+ target mem-> extra specificity to fusion- Exocyst specific to exocytosis, TRAPP I to ER-> Golgi traffic. Exp: golgi tethers re-targeted to mt-> golgi-destined vesicles accum in mt w/out fusing- show ability to bind specific vesicles before fusion.
How tethers recruited to right mem largely ctrled by fam/small GTPases- Rab proteins (Ypt/Sec in yeast). Ypt-protein Sec4 deficient yeast accumulate golgi-derived secretory vesicles-> Sec4 necessary for fusion w/ plasma mem. Rab proteins specific to organelles, e.g., Rab1 ER, Rab4/5 EE, Rab6 Golgi, Rab7 LEs- hence help specify compartment ID. F(x) as molecular switches: GDP-bound form cytosolic, don’t interact trafficking machinery; GTP-bound form extends lipid anchor, allowing to bind mems+ an bind other proteins- Rab effectors (incl motors, enzymes, GEFs, GAPs, tethering factors). GTP hydrolysis, and GD-TP exchange mediated by GEFs (exchange)+ GAPs (GTPase activation). Rabs considered central organisers/ mem traffic- create microdomains/specific protein+ lipid composition which guid traffic. E.g Ees- Rab5 recruits effectors Rabaptin5+ VPS34 (PI(3)Kinase)-> recruitment of EEA1 tether. Vesicles may also remain partly coated+ coats interact target mem tethers-> further specificity.
Membrane fusion experimental approaches: in vitro vesicle fusion assay and yeast genetics
Membrane fusion: exp approaches: synaptic vesicles model system, in vitro mem fusion assay, yeast genetics
In vitro vesicle fusion assay (Rothman+ co) measures transport in Golgi stack by monitoring glycosylation state/ viral protein VSVG. Cells deficient in GlcNAc transferase (medial Golgi, normally modifies VSVG)+ expressing VSVG. Golgi stacks from these cells used as ‘donor’. Stacks from wild type cells not exp VSVG aas ‘acceptor’, w/ all enzymes for ful VSVG glycosylation. Transport between stacks monitored by assaying GlcNAc incorp into VSVG. Transport req adding cytosol+ ATP. Exp showed that COPI coat necessary for intra golgi transport, also ATPase NSF (NEM-sensitive fusion protein)+ alpha-SNAP (soluble NSF attachment protein).
Yeast genetics: NSF (sec18p)+ alpha-SNAP (sec17p) homologues ID’d by schekman’s sec screen – both sec mutants blocked in ER->Golgi transport. Conclude that fusion machinery appears highly conserved between organisms+ diff steps of pathway (hence should also f(x) in synaptic vesicles)
SNAREs and evidence fore them
SNAREs: Rothman isolated protein complexes w/ NSF+ SNAP from brain detergent extracts. Novel components of complex= SNAREs (SNAP receptors), incl synaptic vesicle protein synaptobrevin+ 2 plasma mem proteins syntaxin+ SNAP-25. ‘SNARE hypothesis’- every vesicle carries synaptobrevin-like molecule (v-snare), every target mem a syntaxin-like one (t-snare). Only specific combos fusogenic-> specificity in fusion. Since confirmed by specific SNARE ID. SNAREs=integral mem proteins w/ no/v small luminal domains+ rod-like cytosolic domains. All have conserved helical domain (snare domain). V-snare= single snare molecule. T-snare= complex of either 3 integral mem snare molecules or 1 integral me snare+ a soluble 2-snare domain.
Fusion: SNARE domains interact by forming stable coiled-coil complexes. E release by pairing dives mem fusion. Multiple complexes zip-> 4-helical bundle (trans-SNARE complex- 6 pairings in synaptic vesicle fusion). Complex formation brings vesicle+ target mem together, may also deform mems via integral mem domains. Eventually, lipids fuse via hemi-fused intermediate+ fusion pore
Evidence for SNAREs as minimal machinery necessary for fusion: 2 populations/lysosomes displaying only suitable c/tsnares on surface can fuse- rate v slow, suggesting other factors in vivo (e.g. vesicle tethering) increase efficiency.
Vesicle/SNARE recycling
Recycling: NSF+ SNAP break up SNARE: v+ t need disentangling. SNAP binds SNARE complex, recruits NSF. ATP hydrolysis drives disassembly of SNAREs- E conserved in potential of v+t to bind. After separation, v-snares return to compartment they budded off in inactive state (otherwise would target carrier back to anterograde compartment immediately). Inactivation poorly understood, likely req reg proteins/special adaptors. NB Retrograde carrier containing recycling v-snare has its own v-snare to specify retrograde transport+ also cycle between compartments. Most compartment identifiers= peripheral proteins released from target mem or lipids that can change Pi state. Recycling problem confined to integral mem components.SNARE pairing explains fusion specificity but doesn’t account for it alone. Pairing promiscuous in vitro; snares often appear to decorate compartments homogenously (ish) but fusion often @ ‘hot spots’; EM pics/ nerve terminals treated w/ botulinum+ tetanus toxin (selectively cleave+ inactivate snares) show synaptic vesicles tightly tethered to mem-> specificity @ multiple levels, incl delivery to right place, tethering vesicle+ fusion
Organelle identity and pathways summary
