Phosphorylation-induced conformational changes regulate GGAs 1 and 3 function at the trans-Golgi network.
The GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) are a family of multidomain proteins implicated in protein trafficking between the Golgi and the endosomes. All three GGAs (1, 2, and 3) bind to the mannose 6-phosphate receptor tail via their VHS domains, as well as to the adaptor protein complex-1 via their hinge domains. The latter interaction has been proposed to be important for cooperative packaging of cargo into forming clathrin-coated carriers at the trans-Golgi network. Here we present evidence that GGA1 function is highly regulated by cycles of phosphorylation and dephosphorylation. Cell fractionation showed that the phosphorylated pool of GGA1 resided predominantly in the cytosol and that recruitment onto membranes was associated with dephosphorylation. Okadaic acid inhibition studies and in vitro dephosphorylation assays indicated that dephosphorylation is mediated by a protein phosphatase 2A-like phosphatase. Dephosphorylation of GGA1 induced a change in the conformation to an "open" form as measured by gel filtration and sucrose gradient analyses. This was associated with enhanced binding to ligands because of release of autoinhibition and increased binding to the adaptor protein complex-1 gamma-appendage. A model is proposed for the regulation of GGA1 function at the trans-Golgi network.
Ghosh
Pradipta
P
Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Kornfeld
Stuart
S
D020727:ADP-Ribosylation Factors / Q000737:chemistry*; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry*; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D015151:Immunoblotting; D007313:Insecta; D008024:Ligands; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D021601:trans-Golgi Network / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C407306:GGA adaptor proteins; D008024:Ligands; D013395:Sucrose; D020727:ADP-Ribosylation Factors