Spatial and functional relationship of GGAs and AP-1 in Drosophila and HeLa cells.
The GGAs [Golgi-localised, gamma-ear containing, ARF (ADP ribosylation factor)-binding proteins] and the AP-1 (adaptor protein-1) complex are both adaptors for clathrin-mediated intracellular trafficking, but their relationship to each other is unclear. We have used two complementary systems, HeLa cells and Drosophila Dmel2 cells, to investigate GGA and AP-1 function. Immunoelectron microscopy of endogenous AP-1 and GGA in Dmel2 cells shows that they are predominantly associated with distinct clathrin-coated structures. Depletion of either GGA or AP-1 by RNAi does not affect the incorporation of the other adaptor into clathrin-coated vesicles (CCVs), and the cargo protein GFP-LERP (green fluorescent protein-lysosomal enzyme receptor protein) is lost from CCVs only when both adaptors are depleted. Similar results were obtained using HeLa cells treated with siRNA to deplete all three GGAs simultaneously. AP-1 was still incorporated into CCVs after GGA depletion and vice versa, and both needed to be depleted for a robust inhibition of receptor-mediated sorting of lysosomal hydrolases. In contrast, downregulation of major histocompatibility complex (MHC) class I by HIV-1 Nef, which requires AP-1, was not affected by a triple GGA knockdown. Thus, our results indicate that the two adaptors can function independently of each other.
University of Cambridge, Cambridge Institute for Medical Research, Cambridge CB2 0XY, UK. jh228@cam.ac.uk
Jennifer
J
Hirst
Daniela A
DA
Sahlender
Maja
M
Choma
Rita
R
Sinka
Michael E
ME
Harbour
Michael
M
Parkinson
Margaret S
MS
Robinson
D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D004330:Drosophila / Q000166:cytology* / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014162:Transfection
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D034741:RNA, Small Interfering; D018808:Transcription Factor AP-1; D049452:Green Fluorescent Proteins