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Journal of Biochemistry Advance Access originally published online on March 13, 2008
Journal of Biochemistry 2008 143(6):803-811; doi:10.1093/jb/mvn034
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© 2008 The Japanese Biochemical Society

In vitro Formation of a Novel Type of Membrane Vesicles Containing Dpm1p: Putative Transport Vesicles for Lipid Droplets in Budding Yeast

Yuichi Takeda1,*,{dagger} and Akihiko Nakano1,2

1Molecular Membrane Biology Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa, Wako, Saitama 351-0198 Japan; and 2Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

{dagger}To whom correspondence should be addressed. Tel: 81-6-6879-4142, Fax: 81-6-6879-4137, E-mail: y-takeda{at}biochem.med.osaka-u.ac.jp

Received January 13, 2008; Accepted February 21, 2008


  Abstract

A novel type of membrane vesicles was formed in vitro from microsomes of Saccharomyces cerevisiae, which carries Dpm1p, an enzyme involved in dolichol-sugar synthesis, but not a typical secretory cargo. While COPII vesicles formed in vitro were sedimentable by centrifugation at 200,000gmax for 15 min, the novel vesicles were not. However, they were sedimented by additional centrifugation at the same speed for 1 h. Immunoelectron microscopy showed that the Dpm1p-containing vesicles had small vesicular/saccular structures of around 40–50 nm in diameter. The addition of glycerol-3-phosphate and oleoyl-CoA, substrates for lipid biosynthesis, significantly enhanced the efficiency of vesicle budding in an ATP-dependent fashion. Dpm1p was localized to lipid droplets as well as endoplasmic reticulum. Fluorescence microscopy further showed that Dpm1p-GFP was present in restricted subregions in isolated lipid droplets. The possibility that the vesicles were intermediates from the endoplasmic reticulum to lipid droplets was examined, and their possible role is discussed.

Key Words: Dpm1p, Erg6p, lipid droplet, Saccharomyces cerevisiae, vesicle transport

Abbreviations: ER, endoplasmic reticulum; GFP, (enhanced) green fluorescent protein; GMP-PNP, guanylyl imidodiphosphate; HSP, high-speed pellet; HSS, high-speed supernatant; LD(s), lipid droplet(s); MSS, medium-speed supernatant; RFP, (monomeric) red fluorescent protein

*Present address: Department of Biochemistry, Osaka University Graduate School of Medicine, 5th Floor, Advanced Research Building, Center for Advanced Science and Innovation, Osaka University, 2-1 Yamada-Oka Suita, Osaka 565-0871, Japan


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