Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport.
Cytosolic coat proteins that bind reversibly to membranes have a central function in membrane transport within the secretory pathway. One well-studied example is COPI or coatomer, a heptameric protein complex that is recruited to membranes by the GTP-binding protein Arf1. Assembly into an electron-dense coat then helps in budding off membrane to be transported between the endoplasmic reticulum (ER) and Golgi apparatus. Here we propose and corroborate a simple model for coatomer and Arf1 activity based on results analysing the distribution and lifetime of fluorescently labelled coatomer and Arf1 on Golgi membranes of living cells. We find that activated Arf1 brings coatomer to membranes. However, once associated with membranes, Arf1 and coatomer have different residence times: coatomer remains on membranes after Arf1-GTP has been hydrolysed and dissociated. Rapid membrane binding and dissociation of coatomer and Arf1 occur stochastically, even without vesicle budding. We propose that this continuous activity of coatomer and Arf1 generates kinetically stable membrane domains that are connected to the formation of COPI-containing transport intermediates. This role for Arf1/coatomer might provide a model for investigating the behaviour of other coat protein systems within cells.
Presley
John F
JF
Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Ward
Theresa H
TH
Pfeifer
Andrea C
AC
Siggia
Eric D
ED
Phair
Robert D
RD
Lippincott-Schwartz
Jennifer
J
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A / Q000494:pharmacology; D016466:CHO Cells; D022181:COP-Coated Vesicles / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D020755:Coat Protein Complex I / Q000378:metabolism*; D020756:Coatomer Protein / Q000378:metabolism; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D007425:Intracellular Membranes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics
D020755:Coat Protein Complex I; D020756:Coatomer Protein; D020126:Brefeldin A; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1