ARFGAP2 and ARFGAP3 are essential for COPI coat assembly on the Golgi membrane of living cells.
Coat protein complex I (COPI) vesicles play a central role in the recycling of proteins in the early secretory pathway and transport of proteins within the Golgi stack. Vesicle formation is initiated by the exchange of GDP for GTP on ARF1 (ADP-ribosylation factor 1), which, in turn, recruits the coat protein coatomer to the membrane for selection of cargo and membrane deformation. ARFGAP1 (ARF1 GTPase-activating protein 1) regulates the dynamic cycling of ARF1 on the membrane that results in both cargo concentration and uncoating for the generation of a fusion-competent vesicle. Two human orthologues of the yeast ARFGAP Glo3p, termed ARFGAP2 and ARFGAP3, have been demonstrated to be present on COPI vesicles generated in vitro in the presence of guanosine 5'-3-O-(thio)triphosphate. Here, we investigate the function of these two proteins in living cells and compare it with that of ARFGAP1. We find that ARFGAP2 and ARFGAP3 follow the dynamic behavior of coatomer upon stimulation of vesicle budding in vivo more closely than does ARFGAP1. Electron microscopy of ARFGAP2 and ARFGAP3 knockdowns indicated Golgi unstacking and cisternal shortening similarly to conditions where vesicle uncoating was blocked. Furthermore, the knockdown of both ARFGAP2 and ARFGAP3 prevents proper assembly of the COPI coat lattice for which ARFGAP1 does not seem to play a major role. This suggests that ARFGAP2 and ARFGAP3 are key components of the COPI coat lattice and are necessary for proper vesicle formation.
Kartberg
Fredrik
F
Department of Medical and Clinical Genetics, Institute of Biomedicine, University of Gothenburg, 405 30 Göteborg, Sweden.
Asp
Lennart
L
Dejgaard
Selma Y
SY
Smedh
Maria
M
Fernandez-Rodriguez
Julia
J
Nilsson
Tommy
T
Presley
John F
JF
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D020727:ADP-Ribosylation Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D022181:COP-Coated Vesicles / Q000378:metabolism*; D020755:Coat Protein Complex I / Q000235:genetics / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism
C503240:ARFGAP3 protein, human; D020755:Coat Protein Complex I; C119951:GTF2A1L protein, human; D020690:GTPase-Activating Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C525860:ARFGAP2 protein, human