The role of cargo proteins in GGA recruitment.
Coat proteins are recruited onto membranes to form vesicles that transport cargo from one compartment to another, but the extent to which the cargo helps to recruit the coat proteins is still unclear. Here we have examined the role of cargo in the recruitment of Golgi-localized, gamma-ear-containing, ADP ribosylation factor (ARF)-binding proteins (GGAs) onto membranes in HeLa cells. Moderate overexpression of CD8 chimeras with cytoplasmic tails containing DXXLL-sorting signals, which bind to GGAs, increased the localization of all three GGAs to perinuclear membranes, as observed by immunofluorescence. GGA2 was also expressed at approximately twofold higher levels in these cells because it was degraded more slowly. However, this difference only partially accounted for the increase in membrane localization because there was a approximately fivefold increase in GGA2 associated with crude membranes and a approximately 12-fold increase in GGA2 associated with clathrin-coated vesicles (CCVs) in cells expressing CD8-DXXLL chimeras. The effect of cargo proteins on GGA recruitment was reconstituted in vitro using permeabilized control and CD8-DXXLL-expressing cells incubated with cytosol containing recombinant GGA2 constructs. Together, these results demonstrate that cargo proteins contribute to the recruitment of GGAs onto membranes and to the formation of GGA-positive CCVs.
Hirst
Jennifer
J
Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.
Seaman
Matthew N J
MN
Buschow
Sonja I
SI
Robinson
Margaret S
MS
D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism*; D033961:Adaptor Protein Complex 1 / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D016827:CD8 Antigens / Q000235:genetics / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D034622:RNA Interference; D017527:Receptor, IGF Type 2 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D033961:Adaptor Protein Complex 1; D033942:Adaptor Proteins, Vesicular Transport; D016827:CD8 Antigens; C407306:GGA adaptor proteins; C405944:GGA2 protein, human; D021382:Protein Sorting Signals; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020126:Brefeldin A; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D020727:ADP-Ribosylation Factors