Roles for alpha(2)p24 and COPI in endoplasmic reticulum cargo exit site formation.
A two-step reconstitution system for the generation of ER cargo exit sites from starting ER-derived low density microsomes (LDMs; 1.17 g/cc) is described. The first step is mediated by the hydrolysis of Mg(2+)ATP and Mg(2+)GTP, leading to the formation of a transitional ER (tER) with the soluble cargo albumin, transferrin, and the ER-to-Golgi recycling membrane proteins alpha(2)p24 and p58 (ERGIC-53, ER-Golgi intermediate compartment protein) enriched therein. Upon further incubation (step two) with cytosol and mixed nucleotides, interconnecting smooth ER tubules within tER transforms into vesicular tubular clusters (VTCs). The cytosolic domain of alpha(2)p24 and cytosolic COPI coatomer affect VTC formation. This is deduced from the effect of antibodies to the COOH-terminal tail of alpha(2)p24, but not of antibodies to the COOH-terminal tail of calnexin on this reconstitution, as well as the demonstrated recruitment of COPI coatomer to VTCs, its augmentation by GTPgammaS, inhibition by Brefeldin A (BFA), or depletion of beta-COP from cytosol. Therefore, the p24 family member, alpha(2)p24, and its cytosolic coat ligand, COPI coatomer, play a role in the de novo formation of VTCs and the generation of ER cargo exit sites.
Lavoie
C
C
Département de Pathologie et Biologie Cellulaire, Faculté de Médecine, Université de Montréal, Québec, Canada H3C 3J7.
Paiement
J
J
Dominguez
M
M
Roy
L
L
Dahan
S
S
Gushue
J N
JN
Bergeron
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000418:Albumins / Q000378:metabolism; D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D020756:Coatomer Protein; D003600:Cytosol / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008099:Liver / Q000166:cytology; D037241:Mannose-Binding Lectins; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D014168:Transferrin / Q000378:metabolism
D000418:Albumins; D002135:Calcium-Binding Proteins; D020756:Coatomer Protein; C501458:Lman1 protein, rat; D037241:Mannose-Binding Lectins; D008565:Membrane Proteins; D008869:Microtubule-Associated Proteins; D014168:Transferrin; D037281:Calnexin; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate