#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. Rec

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

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 vi

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 tru

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

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

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