Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

doi:10.1083/jcb.200412003

Published online 22 February 2005. doi:10.1083/jcb.200412003
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 168, Number 5, 747-759

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Article

Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

Sergey N. Zolov and Vladimir V. Lupashin

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR 72205

Correspondence to Vladimir Lupashin: vvlupashin{at}uams.edu

The conserved oligomeric Golgi (COG) complex is an evolutionarilyconserved multi-subunit protein complex that regulates membranetrafficking in eukaryotic cells. In this work we used shortinterfering RNA strategy to achieve an efficient knockdown (KD)of Cog3p in HeLa cells. For the first time, we have demonstratedthat Cog3p depletion is accompanied by reduction in Cog1, 2,and 4 protein levels and by accumulation of COG complex-dependent(CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgiglycoprotein GPP130. Some of these CCD vesicles appeared tobe vesicular coat complex I (COPI) coated. A prolonged blockin CCD vesicles tethering is accompanied by extensive fragmentationof the Golgi ribbon. Fragmented Golgi membranes maintained theirjuxtanuclear localization, cisternal organization and are competentfor the anterograde trafficking of vesicular stomatitis virusG protein to the plasma membrane. In a contrast, Cog3p KD resultedin inhibition of retrograde trafficking of the Shiga toxin.Furthermore, the mammalian COG complex physically interactswith GS28 and COPI and specifically binds to isolated CCD vesicles.

Abbreviations used in this paper: CCD, COG complex-dependent;COG, conserved oligomeric Golgi; COPI, vesicular coat complexI; GalNAc-T2, N-acetylgalactosaminyltransferase-2; GalT-GFP,GFP-tagged ß 1,4-galactosyltransferase; IF, immunofluorescence;IP, immunoprecipitation; KD, knockdown; PDI, protein disulphideisomerase; PNS, post-nuclear supernatant; siRNA, short interferingRNA; VSVG, vesicular stomatitis virus G protein; WB, Westernblot.

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