The GGAs (Golgi-localized, γ ear–containing, ADP ribosylation factor–binding proteins) are multidomain proteins implicated in protein trafficking between the Golgi and endosomes. We examined whether the three mammalian GGAs act independently or together to mediate their functions. Using cryo-immunogold electron microscopy, the three GGAs were shown to colocalize within coated buds and vesicles at the trans-Golgi network (TGN) of HeLa cells. In vitro binding experiments revealed multidomain interactions between the GGAs, and chemical cross-linking experiments demonstrated that GGAs 1 and 2 form a complex on Golgi membranes. RNA interference of each GGA resulted in decreased levels of the other GGAs and their redistribution from the TGN to cytosol. This was associated with impaired incorporation of the cation-independent mannose 6-phosphate receptor into clathrin-coated vesicles at the TGN, partial redistribution of the receptor to endosomes, and missorting of cathepsin D. The morphology of the TGN was also altered. These findings indicate that the three mammalian GGAs cooperate to sort cargo and are required for maintenance of TGN structure.
Mammalian GGAs act together to sort mannose 6-phosphate receptors
P. Ghosh's present address is University of California, San Diego Medical Center, Department of Medicine, 200 West Arbor Drive, San Diego, CA 92103-8422.
Abbreviations used in this paper: AP-1, adaptor protein 1; ARF, ADP ribosylation factor; β-GalT, β-galactosyltransferase; CCV, clathrin-coated vesicle; CI-MPR, cation-independent MPR; DTSSP, 3,3′-dithiobis[sulfosuccinimidylpropionate]; EEA1, early endosomal antigen 1; GAT, GGA and Tom; GGA, Golgi-localized, γ ear–containing, ADP ribosylation factor–binding protein; MPR, mannose 6-phosphate receptor; RNAi, RNA interference; siRNA, small interfering RNA; VHS, Vps, Hrs, and STAM.
Pradipta Ghosh, Janice Griffith, Hans J. Geuze, Stuart Kornfeld; Mammalian GGAs act together to sort mannose 6-phosphate receptors . J Cell Biol 24 November 2003; 163 (4): 755–766. doi: https://doi.org/10.1083/jcb.200308038
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