#vesicle transport

Protein Sorting and Trafficking

Which of the following does NOT require clathrin-coated vesicles?

Coat identity determines vesicle origin and destination providing directional logic to secretory pathway. COPII composed of Sar1 initiating curvature, inner adaptor Sec23-Sec24 binding cargo export signals, outer cage Sec13-Sec31 polymerizing into cuboctahedron operates exclusively at ER exit sites producing vesicles moving toward ERGIC and cis-Golgi carrying newly synthesized secretory proteins bearing ER exit motifs DXE, LXXLE. COPI composed of ARF1-GTP and heptameric coatomer operates reciprocally at Golgi rims and ERGIC retrieving material backward including KKXX membrane proteins and KDEL receptor bound luminal chaperones. Clathrin triskelia with heterotetrameric adaptors AP1 at TGN, AP2 at plasma membrane, GGAs and AP3 operates at later stations where PI4P or PI(4,5)P2 and ARF family GTPases recruit adaptors: TGN to late endosomes for mannose-6-phosphate receptor bound hydrolases, plasma membrane to early endosomes for transferrin uptake, and endosomes to lysosomes. Thus ER to Golgi uniquely COPII dependent mechanistically independent of clathrin lattice. siRNA of clathrin heavy chain blocks transferrin endocytosis and cathepsin D sorting but not ER export of temperature-sensitive VSV-G, while Sec24B depletion arrests ER exit confirming distinct machinery division and functional separation.

Ref: Alberts et al., MBC: COPII ER-to-Golgi transport does not require clathrin coats.

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Protein Sorting and Trafficking

What signal is required for COPII-mediated ER-to-Golgi transport?

Selective inclusion of membrane cargo into COPII vesicles instead of bulk flow relies on export signals recognized directly by Sec24 adaptor subunit. Well-characterized ER export code is di-acidic DXE motif positioned four to ten residues downstream of transmembrane domain in cytosolic C-terminal tails of many type I proteins like VSV-G, Sys1, ERGIC-53, SNAREs Bet1, Sec22. Motif consists of Asp-X-Glu where both acidic side chains fit basic pockets formed by arginine and lysine in Sec24 B-site; upstream hydrophobic aromatic residues strengthen interaction. Crystal structures show Sec24C and Sec24D isoforms especially adept binding DXE while Sec24A-B prefer IxM signals. Mutation of DXE to AXA drastically lowers affinity slowing ER exit five to ten fold causing ER retention and degradation. KKXX at extreme C-terminus binds alpha beta prime COP I subunits for Golgi-to-ER retrieval opposite direction. Tyrosine-based YXXPhi recognized by mu subunit of AP complexes for clathrin-mediated TGN to endosome and plasma membrane endocytosis, NPXY recognized by PTB domains of Dab2 and ARH for LDL receptor uptake. Thus DXE uniquely encodes ER exit into COPII for anterograde traffic ensuring efficient export.

Ref: Lodish et al., MCB: DXE di-acidic motif recognized by Sec24 for COPII ER exit.

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Protein Sorting and Trafficking

Which of the following regulates vesicle docking before fusion?

Before trans-SNARE zippering drives bilayer merger vesicles must be captured near correct target ensuring compartment identity maintained despite promiscuous SNAREs. Rab GTPases provide master specificity for docking stage. Active GTP-bound form on vesicle or target exposes switch regions binding effectors. Examples: Rab1-GTP recruits p115 and GM130 at ER-to-Golgi interface forming extended coiled-coil tether over 200 nm, Rab5-GTP recruits EEA1 and Rabenosyn-5 containing FYVE domains binding PI3P plus alpha helical tether, Rab6 recruits GCC185, Rab7 recruits HOPS complex VPS11-18-39-41. This physical bridging reduces dimensionality searching, proofreads identity, increases local concentration promoting cognate SNARE engagement. Clathrin forms coats for sorting at plasma membrane and TGN, alpha-mannosidase trims N-glycans in Golgi, and SNARE disassembly factors NSF and alpha-SNAP act post-fusion regenerating SNAREs. Rab conversion mediated by GEFs Mon1-Ccz1 and GAPs ensures directionality; Rab5 to Rab7 conversion guides endosome maturation early to late. Ablation of Rab or tether leads to mistargeting and vesicle accumulation despite intact SNAREs present and functional.

Ref: Alberts et al., MBC: Rab GTPases regulate vesicle docking before SNARE-mediated fusion.

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Protein Sorting and Trafficking

What is the function of the SNARE complex?

Vesicle fusion fidelity beyond tethering relies on SNARE proteins providing thermodynamic driver for bilayer merger overcoming hydration repulsion. Vesicle membranes contain R-SNAREs like synaptobrevin VAMP2 with single motif and C-terminal anchor, while target membranes harbor Q-SNAREs syntaxin family with Habc regulatory domain and SNAP-25 family contributing two motifs anchored by palmitoylation. When opposed membranes brought within few nanometers by Rab tethers and multisubunit complexes, oppositely oriented SNARE motifs assemble as parallel four-helix coiled coil from N-terminus to C-terminus zippering releasing substantial free energy. Formation pulls membranes into point contact inducing hemifusion stalk then fusion pore opening delivering content efficiently. Specificity arises from compatible QabcR combinations respecting ionic central layer; neuronal exocytosis uses syntaxin1-SNAP25-VAMP2, ER-Golgi uses Sed5-membrin-Bet1-Bos1-Sec22, endosome uses syntaxin7-8-Vti1b-VAMP8. They do not mediate budding or ATP hydrolysis or clathrin recruitment; those involve coats and GTPases. After fusion cis complex awaits disassembly by NSF ATPase AAA family for reuse throughout secretory pathway.

Ref: Jahn & Scheller, Nat Rev Mol Cell Biol 2006: SNARE four-helix bundle drives membrane fusion.

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Protein Sorting and Trafficking

COPII vesicles mediate transport from:

ER export mediated by COPII coats discovered through sec mutants in yeast by Schekman. At ER exit sites devoid of ribosomes, integral GEF Sec12 converts Sar1 GDP to GTP causing amphipathic helix insertion into ER membrane inducing curvature and recruiting inner coat Sec23-Sec24 heterodimer. Sec24 isoforms provide multiple cargo binding sites recognizing di-acidic DXE, di-hydrophobic, proline-rich and arginine motifs concentrating transmembrane and soluble cargo via receptors Erv29, Erv14, Surf4 and Erv29. Outer coat Sec13-Sec31 heterotetramer forms cage driving deformation and budding producing 60-80 nm vesicles that uncoat after Sar1 hydrolysis mediated by Sec23 GAP activity. Vesicles fuse forming ERGIC then cis-Golgi mediated by SNAREs Sed5, Bet1. COPI drives opposite Golgi-to-ER retrieval, clathrin operates at TGN and plasma membrane for lysosomal sorting and endocytosis. Temperature-sensitive Sar1 mutants block secretion trapping cargo in ER demonstrating COPII essential for anterograde ER to Golgi leg conserved from yeast to human and plants needing precise export code.

Ref: Alberts et al., MBC, Chapter 13: COPII vesicles mediate ER to Golgi anterograde transport.

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Protein Sorting and Trafficking

What is the primary function of Rab proteins in vesicle transport?

Rab small GTPases orchestrate specificity along endomembrane system as largest branch of Ras superfamily cycling between cytosol and membrane. GDP-bound Rabs kept soluble by guanine nucleotide dissociation inhibitor GDI that shields geranylgeranyl tails. Upon recruitment by specific guanine nucleotide exchange factors TRAPP, DENND, Mon1-Ccz1 at target organelles, they exchange GDP for GTP exposing N-terminal amphipathic helices and prenyl anchors firmly embedding in bilayer. GTP conformation exposes switch regions binding diverse effectors: long coiled-coil golgins like GM130, p115, tethering complexes CORVET, HOPS, Dsl1, GARP, TRAPPII, and motors kinesin, dynein, myosin Vb for movement along microtubules and actin. Effectors mediate vesicle capture at up to 200 nm bringing vesicles close for SNARE pairing. After fusion, TBC domain GAPs stimulate GTP hydrolysis returning Rab to GDP and GDI extraction for another cycle. Humans encode over 60 Rabs marking distinct compartments: Rab1 ER-Golgi, Rab5 early endosomes, Rab7 late, Rab11 recycling. They do not directly fuse membranes nor hydrolyze ATP nor degrade cargo.

Ref: Alberts et al., MBC, Chapter 13: Rab GTPases as vesicle tethering and targeting regulators.

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Protein Sorting and Trafficking

In the vesicle transport model, how are Golgi-resident proteins returned to their compartments?

Golgi organization is explained by cisternal maturation model where new cis cisternae arise from fusion of ER-derived COPII vesicles at ERGIC and progressively mature into trans cisternae carrying secretory cargo forward toward trans-Golgi network. Resident processing enzymes, including cis mannosidases, medial GlcNAc transferases and trans sialyltransferases, cannot move forward with cargo otherwise compartmental polarity would be lost and glycosylation random. To preserve sequential enzyme gradients, transmembrane residents are selectively extracted into COPI-coated vesicles that transport retrogradely to younger cisternae. Cytosolic tails expose signals recognized by adaptors such as Vps74, GOLPH3 and COG complex concentrating enzymes into COPI buds. Live imaging in yeast shows COPI vesicles carrying medial mannosidase II backward while cargo remains lumenal and anterograde carriers move forward. Anterograde vesicle shuttle models propose stable cisternae, but COPI mutants and Rab effectors and tether assays demonstrate retrograde return essential for maturation, not degradation, lateral diffusion, or default secretion pathway maintaining fidelity and polarity.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 13: Cisternal maturation and COPI retrograde Golgi traffic.

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Protein Sorting and Trafficking

What is the main function of Rab GTPases in protein trafficking?

Rab family is largest branch of Ras superfamily, more than sixty members in mammals, each localized to specific organelle defining compartment identity and regulating sequential vesicle traffic steps. C-terminal cysteine geranylgeranylation anchors Rab to cytosolic face of membranes. In active GTP-bound conformation, Rab recruits diverse effectors: tethering complexes EEA1 for Rab5 early endosomes, GARP and golgins for retrograde traffic, exocyst for Rab11 recycling vesicles, and motor adaptors linking vesicles to kinesin for anterograde or dynein-dynactin for retrograde movement along microtubules. Guanine exchange factors like Rabex-5 activate Rabs on designated membranes, while RabGAPs containing TBC domain stimulate hydrolysis converting to GDP form releasing effectors and allowing extraction by GDI for recycling. This cycle ensures specificity so COPII vesicles from ER fuse only with cis-Golgi, not lysosomes, and provides timing for endosomal maturation Rab5 to Rab7 conversion. Unlike importin transporting through nuclear pores or kinases phosphorylating SNAREs to regulate fusion, Rab orchestrates upstream vesicle targeting, motility and tethering coordinated with SNARE-mediated final fusion ensuring fidelity of trafficking pathways and organelle biogenesis.

Ref: Zerial & McBride, Nature Rev Mol Cell Biol 2: 2001, Rab GTPases Control Vesicle Targeting.

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