#membrane trafficking

Protein Sorting and Trafficking

Which protein is required for vesicle scission during receptor-mediated endocytosis?

Final separation stage clathrin-coated pit formation yielding free vesicle requires enzymatic fission beyond coat polymerization. Clathrin assembly with adaptor AP2 and BAR proteins epsin, amphiphysin, endophilin generates deeply invaginated bud connected plasma membrane narrow tubule neck diameter around 20 nm bilayer. Separation demands mechanical constriction cutting neck. Specialist protein dynamin large 96 kDa GTPase family founding member N-terminal GTPase domain, middle stalk, PH domain binding PI(4,5)P2 enriched site, GED and PRD binding SH3, polymerizes helical collar around neck. Recruitment mediated curvature-sensing BAR proteins generating curvature. Stoichiometry about 13 dimers per turn aligns G domains efficient hydrolysis. GTP binding tightens helix reducing lumen radius; cooperative hydrolysis drives constriction twisting shear breaking membrane hemi-fission intermediate releasing vesicle. Clathrin provides structural cage not enzymatic fission; Rab GTPases regulate downstream tethering early endosome via EEA1, SNAREs mediate subsequent fusion after uncoating. Temperature-sensitive shibire mutant drosophila dynamin blocks synaptic vesicle recycling restrictive temperature trapping pits long collars demonstrating indispensable scission function receptor-mediated endocytosis essential cellular uptake.

Ref: Kaksonen & Roux, Nat Rev Mol Cell Biol: Dynamin scission in clathrin-mediated endocytosis.

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Protein Sorting and Trafficking

The acidic pH in the late endosome causes:

Endosomal maturation involves Rab conversion progressive luminal acidification orchestrated by vacuolar V-type ATPase multisubunit rotary pump hydrolyzing ATP pumping protons generating pH early endosome 6.0-6.5 late 5.5-6.0 lysosome 4.5-5.0. Gradient critical sorting function beyond hydrolysis activation many ligand-receptor complexes exhibit pH dependent affinity due histidine protonation altering charge complementarity. Examples: LDL separates from LDL receptor pH below 6 releasing particle for lysosomal degradation while receptor recycles via retromer, mannose-6-phosphate receptors release lysosomal enzymes upon protonation binding pocket, transferrin releases Fe3+ upon protonation carbonate while apotransferrin remains bound retained high affinity acidic pH. Dissociation allows physical separation receptors segregate narrow recycling tubules via SNX while liberated ligands continue lysosome catabolism. Without acidification bafilomycin or weak base ammonium chloride pH rise sorting fails LDL remains bound preventing recycling and signaling. Clathrin assembly independent acidity, proteasomal degradation cytosolic, mitochondria fusion unrelated pH-triggered endosomal dissociation ensuring efficient cargo segregation and receptor reuse essential homeostasis.

Ref: Maxfield & McGraw, Nat Rev Mol Cell Biol: Endosomal acidification dissociates receptor-ligand complexes.

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Protein Sorting and Trafficking

Which of the following is TRUE about AP (adapter protein) complexes?

Heterotetrameric adapter protein complexes link cargo sorting signals to clathrin and other coats decoding destinations. AP2 composed of alpha, beta2, mu2, sigma2 about 300 kDa localizes exclusively to plasma membrane where alpha appendage ear binds PIP2 and accessory proteins Eps15 and epsin. Mu2 middle domain recognizes YXXPhi tyrosine signals where tyrosine sits in deep hydrophobic pocket and phi bulky hydrophobic; affinity enhanced by phosphorylation at Thr156 by AAK1 kinase upon membrane recruitment. Dileucine [DE]XXXL[LI] also recognized. Transferrin receptor YTRF, LDL receptor FXNPXY via co-adaptor ARH linking to AP2, and many signaling receptors use this for rapid internalization forming pits 0.5 percent surface. AP1 at TGN and endosomes contains gamma adaptin and mediates TGN to endosome traffic, not Golgi to ER which uses COPI dilysine. AP3 delivers to lysosomes and lysosome-related organelles melanosomes via tyrosine and dileucine; COPII formation depends on Sec24 not AP3. AP complexes use clathrin or related coats; interaction with cargo tails not SNAREs defines sorting specificity bridging membrane, signal, and coat.

Ref: Robinson, Trends Cell Biol 2015: AP2 adaptor complex in clathrin-mediated endocytosis at plasma membrane.

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Protein Sorting and Trafficking

What is the main function of ARF-GTP in vesicle formation?

Budding of AP1, AP3 and GGA clathrin-coated vesicles at trans-Golgi network and endosomes requires activated ARF1 small GTPase cycling between GDP and GTP states. ARF1-GDP cytosolic bound to GDI-like factor; activation by large ARF GEFs BIG1-BIG2 at TGN and GBF1 at Golgi exchange GDP for GTP upon membrane recruitment mediated by HDS domains sensing curvature and lipid composition. ARF-GTP undergoes structural rearrangement exposing N-terminal myristoylated amphipathic helix inserting into outer leaflet and effector binding interface. Effectors include heterotetrameric AP1 via gamma subunit trunk, AP3 via delta subunit, AP4, and monomeric GGAs via GAT domain, plus activation of PI4KIIIbeta generating PI4P enhancing adaptor affinity and phospholipase D producing phosphatidic acid promoting negative curvature. Coordinated recruitment concentrates lysosomal cargo such as mannose-6-phosphate receptors bearing acidic cluster dileucine motifs. ARF does not hydrolyze ATP; ATP independent. Scission involves dynamin-family or Arf GAP-induced curvature together with BAR proteins, not SNARE scaffolding. Hydrolysis by ARF GAP1 later triggers partial uncoating for fusion competence and recycling.

Ref: Lodish et al., MCB Chapter 14: ARF1-GTP recruits AP1/GGA to initiate clathrin coat at TGN.

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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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