Distinct and overlapping roles for AP-1 and GGAs revealed by the "knocksideways" system.
Although adaptor protein complex 1 (AP-1) and Golgi-localized, γ ear-containing, ADP-ribosylation factor-binding proteins (GGAs) are both adaptors for clathrin-mediated intracellular trafficking, the pathways they mediate and their relationship to each other remain open questions. To tease apart the functions of AP-1 and GGAs, we rapidly inactivated each adaptor using the "knocksideways" system and then compared the protein composition of clathrin-coated vesicle (CCV) fractions from control and knocksideways cells. The AP-1 knocksideways resulted in a dramatic and unexpected loss of GGA2 from CCVs. Over 30 other peripheral membrane proteins and over 30 transmembrane proteins were also depleted, including several mutated in genetic disorders, indicating that AP-1 acts as a linchpin for intracellular CCV formation. In contrast, the GGA2 knocksideways affected only lysosomal hydrolases and their receptors. We propose that there are at least two populations of intracellular CCVs: one containing both GGAs and AP-1 for anterograde trafficking and another containing AP-1 for retrograde trafficking. Our study shows that knocksideways and proteomics are a powerful combination for investigating protein function, which can potentially be used on many different types of proteins.
University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. jh228@cam.ac.uk
Jennifer
J
Hirst
Georg H H
GH
Borner
Robin
R
Antrobus
Andrew A
AA
Peden
Nicola A
NA
Hodson
Daniela A
DA
Sahlender
Margaret S
MS
Robinson
D033961:Adaptor Protein Complex 1 / Q000235:genetics / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002966:Clathrin / Q000172:deficiency / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011485:Protein Binding; D050683:R-SNARE Proteins / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering; D050600:SNARE Proteins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D033961:Adaptor Protein Complex 1; D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; C407306:GGA adaptor proteins; D050683:R-SNARE Proteins; C097227:RABEP1 protein, human; D034741:RNA, Small Interfering; D050600:SNARE Proteins; C476456:VAMP4 protein, human; D033921:Vesicular Transport Proteins