Discrete determinants in ArfGAP2/3 conferring Golgi localization and regulation by the COPI coat.
From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein-protein and protein-lipid interactions to promote GTP hydrolysis in Arf1-GTP.
Kliouchnikov
Lena
L
Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
Bigay
Joëlle
J
Mesmin
Bruno
B
Parnis
Anna
A
Rawet
Moran
M
Goldfeder
Noga
N
Antonny
Bruno
B
Cassel
Dan
D
D017607:Aluminum Compounds / Q000494:pharmacology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D015704:CD4 Antigens / Q000378:metabolism; D020755:Coat Protein Complex I / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology; D020690:GTPase-Activating Proteins / Q000737:chemistry* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D013329:Structure-Activity Relationship
D017607:Aluminum Compounds; D015704:CD4 Antigens; D020755:Coat Protein Complex I; D020690:GTPase-Activating Proteins; D008081:Liposomes; D050505:Mutant Proteins; D005459:Fluorides; C032311:aluminum fluoride