Binding partners for the COOH-terminal appendage domains of the GGAs and gamma-adaptin.
The adaptor appendage domains are believed to act as binding platforms for coated vesicle accessory proteins. Using glutathione S-transferase pulldowns from pig brain cytosol, we find three proteins that can bind to the appendage domains of both the AP-1 gamma subunit and the GGAs: gamma-synergin and two novel proteins, p56 and p200. p56 elicited better antibodies than p200 and was generally more tractable. Although p56 and gamma-synergin bind to both GGA and gamma appendages in vitro, immunofluorescence labeling of nocodazole-treated cells shows that p56 colocalizes with GGAs on TGN46-positive membranes, whereas gamma-synergin colocalizes with AP-1 primarily on a different membrane compartment. Furthermore, in AP-1-deficient cells, p56 remains membrane-associated whereas gamma-synergin becomes cytosolic. Thus, p56 and gamma-synergin show very strong preferences for GGAs and AP-1, respectively, in vivo. However, the GGA and gamma appendages share the same fold as determined by x-ray crystallography, and mutagenesis reveals that the same amino acids contribute to their binding sites. By overexpressing wild-type GGA and gamma appendage domains in cells, we can drive p56 and gamma-synergin, respectively, into the cytosol, suggesting a possible mechanism for selectively disrupting the two pathways.
Lui
Winnie W Y
WW
Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, United Kingdom.
Collins
Brett M
BM
Hirst
Jennifer
J
Motley
Alison
A
Millar
Caroline
C
Schu
Peter
P
Owen
David J
DJ
Robinson
Margaret S
MS
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033961:Adaptor Protein Complex 1; D033982:Adaptor Protein Complex gamma Subunits / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009154:Mutation; D017434:Protein Structure, Tertiary
D033961:Adaptor Protein Complex 1; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D002352:Carrier Proteins; C407306:GGA adaptor proteins; C121510:SYNRG protein, human; C524122:p56 trans-Golgi network accessory protein, human; D020727:ADP-Ribosylation Factors