Multiple phosphorylation events regulate the subcellular localization of GGA1.
GGAs comprise a family of Arf-dependent coat proteins or adaptors that regulate vesicle traffic from the trans-Golgi network (TGN). GGAs bind activated Arf, cargo, and additional components necessary for vesicle budding through interactions with their four functional domains: VHS, GAT, hinge, and GAE. We identified three sites of phosphorylation in GGA1 by tandem mass spectrometry: S268 and T270 in the GAT domain and S480 in the hinge. Expression of HA-GGA1 in mammalian cells and comparison to endogenous GGA1 confirmed their localization to late Golgi compartments. In contrast, mutations that mimic the phosphoprotein (HA-GGA1[S268D] or HA-GGA1[T270D]) at either of the sites in the GAT domain caused a decrease in the colocalization with markers of the Golgi and TGN and an increase in puncta in cytoplasm. Quantitative comparisons of the extent of colocalization of GGA1 proteins with the known components of GGA1 vesicles revealed that the composition of those markers tested in HA-GGA1[S268D] and HA-GGA1[T270D] vesicles were indistinguishable from those of HA-GGA1 vesicles. We conclude that phosphorylation of the GAT domain can stabilize the coat proteins bound and thus regulate the rate of coat protein dissociation.
McKay
Melissa M
MM
Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Rd, Atlanta, GA 30322-3050, USA.
Kahn
Richard A
RA
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D015415:Biomarkers; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D004268:DNA-Binding Proteins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012694:Serine / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D015415:Biomarkers; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C407306:GGA adaptor proteins; D033921:Vesicular Transport Proteins; D013912:Threonine; D012694:Serine; D020727:ADP-Ribosylation Factors; D020691:rab GTP-Binding Proteins