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Vol. 18, Issue 12, 4762-4771, December 2007

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Golgi-bound Rab34 Is a Novel Member of the Secretory Pathway

Neil M. Goldenberg^*, Sergio Grinstein, and Mel Silverman

*Institute of Medical Science and Department of Medicine, University of Toronto, Toronto, ON, Canada, M5S 1A8; and Program in Cell Biology, Hospital for Sick Children, Toronto, ON, Canada, M5G 2C4

Submitted November 6, 2006; Revised September 5, 2007; Accepted September 11, 2007
Monitoring Editor: Jennifer Lippincott-Schwartz

Golgi-localized Rab34 has been implicated in repositioning lysosomes and activation of macropinocytosis. Using HeLa cells, we undertook a detailed investigation of Rab34 involvement in intracellular vesicle transport. Immunoelectron microscopy and immunocytochemistry confirmed that Rab34 is localized to the Golgi stack and that active Rab34 shifts lysosomes to the cell center. Contrary to a previous report, we found that Rab34 is not concentrated at membrane ruffles and is not involved in fluid-phase uptake. Also, Rab34-induced repositioning of lysosomes does not affect mannose 6-phosphate receptor trafficking. Most strikingly, HeLa cells depleted of Rab34 by transfection with dominant-negative Rab34 or after RNA interference, failed to transport the temperature-sensitive vesicular stomatitis virus G-protein (VSVG) fused to green fluorescent protein (VSVG-GFP) from the Golgi to the plasma membrane. Transfection with mouse Rab34 rescued this defect. Using endogenous major histocompatibility complex class I (MHCI) as a marker, an endoglycosidase H resistance assay showed that endoplasmic reticulum (ER) to medial Golgi traffic remains intact in knockdown cells, indicating that Rab34 specifically functions downstream of the ER. Further, brefeldin A treatment revealed that Rab34 effects intra-Golgi transport, not exit from the trans-Golgi network. Collectively, these results define Rab34 as a novel member of the secretory pathway acting at the Golgi.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Mel Silverman (melvin.silverman{at}utoronto.ca).

Abbreviations used: BFA, brefeldin A; CA-GFP-Rab34, GFP-tagged constitutively active Rab34; DN-GFP-Rab34, GFP-tagged dominant-negative Rab34; EndoH, endoglycosidase H; GFP, green fluorescent protein; HRas-Tail-RFP, RFP-tagged tail of H-Ras; MHC, major histocompatibility complex; M6PR, mannose 6-phosphate receptor; PI4P, phosphoinositol 4-phosphate; PKD, protein kinase D; RFP, red fluorescent protein; TGN, trans-Golgi network; TPA, 2-O-tetradecanoyl-phorbol 13-acetate; VSVG, vesicular stomatitis virus G protein; WGA, wheat germ agglutinin; wt-GFP-Rab34, GFP-tagged wild-type Rab34.