Secretory cargo regulates the turnover of COPII subunits at single ER exit sites.
The COPII coat complex mediates the formation of transport carriers at specialized sites of the endoplasmic reticulum (ERES). It consists of the Sar1p GTPase and the Sec23/24p and the Sec13/31p subcomplexes . Both stimulate the GTPase activity of Sar1p , which itself triggers coat disassembly. This built-in GAP activity makes the COPII complex in principle unstable and raises the question of how sufficient stability required for cargo capture and carrier formation is achieved. To address this, we analyzed COPII turnover at single ERES in living cells. The half times for Sar1p, Sec23p, and Sec24p turnover are 1.1, 3.7, and 3.9 s, respectively. Decreasing the amount of transport-competent cargo in the endoplasmic reticulum accelerates turnover of the Sec23/24p and slows down that of Sar1p. A mathematical model of COPII membrane turnover that reproduces the experimental in vivo FRAP kinetics and is consistent with existing in vitro data predicts that Sec23/24p remains membrane associated even after GTP hydrolysis by Sar1p for a duration that is strongly increased by the presence of cargo. We conclude that secretory cargo retains the COPII complex on membranes, after Sar1p release has occurred, and prevents premature disassembly of COPII during cargo sorting and transport carrier formation.
Forster
Rebecca
R
Cell Biology and Biophysics Unit, EMBL, 69117 Heidelberg, Germany.
Weiss
Matthias
M
Zimmermann
Timo
T
Reynaud
Emmanuel G
EG
Verissimo
Fatima
F
Stephens
David J
DJ
Pepperkok
Rainer
R
D000818:Animals; D022181:COP-Coated Vesicles / Q000187:drug effects / Q000378:metabolism*; D002522:Chlorocebus aethiops; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D020559:Monomeric GTP-Binding Proteins / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D014709:Vero Cells; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D021122:Protein Subunits; D011500:Protein Synthesis Inhibitors; D033921:Vesicular Transport Proteins; D003513:Cycloheximide; C493083:SAR1A protein, human; C513282:SAR1B protein, human; D020559:Monomeric GTP-Binding Proteins