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Published 24 November 2003. doi:10.1083/jcb.200308038
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© The Rockefeller University Press, 0021-9525/2003/11/755 $8.00
The Journal of Cell Biology, Volume 163, Number 4, 755-766

Article

Mammalian GGAs act together to sort mannose 6-phosphate receptors



Pradipta Ghosh1, Janice Griffith2, Hans J. Geuze2 and Stuart Kornfeld1

1 Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110
2 Department of Cell Biology, University Medical Center and Institute of Biomembranes, Utrecht University, 3584 CX Utrecht, Netherlands

Address correspondence to Stuart Kornfeld, Washington University School of Medicine, Dept. of Internal Medicine, 660 S. Euclid Ave., Box 8125, St. Louis, MO 63110. Tel.: (314) 362-8803. Fax: (314) 362-8826. email: skornfel{at}im.wustl.edu

The GGAs (Golgi-localized, {gamma} 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.

Key Words: trans-Golgi network; clathrin-coated vesicle; adaptor protein 1; siRNA; cryo-immunogold EM

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; ß-GalT, ß-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, {gamma} ear–containing, ADP ribosylation factor–binding protein; MPR, mannose 6-phosphate receptor; RNAi, RNA interference; siRNA, small interfering RNA; VHS, Vps, Hrs, and STAM.


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