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Originally published as MBC in Press, 10.1091/mbc.E05-05-0452 on September 29, 2005

Vol. 16, Issue 12, 5592-5609, December 2005

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Defects in Structural Integrity of Ergosterol and the Cdc50p-Drs2p Putative Phospholipid Translocase Cause Accumulation of Endocytic Membranes, onto Which Actin Patches Are Assembled in Yeast

Takuma Kishimoto, Takaharu Yamamoto, and Kazuma Tanaka

Division of Molecular Interaction, Institute for Genetic Medicine, Hokkaido University Graduate School of Medicine, N15 W7, Kita-ku, Sapporo 060-0815, Japan

Submitted May 23, 2005; Revised August 24, 2005; Accepted September 15, 2005
Monitoring Editor: Howard Riezman

Specific changes in membrane lipid composition are implicated in actin cytoskeletal organization, vesicle formation, and control of cell polarity. Cdc50p, a membrane protein in the endosomal/trans-Golgi network compartments, is a noncatalytic subunit of Drs2p, which is implicated in translocation of phospholipids across lipid bilayers. We found that the cdc50{Delta} mutation is synthetically lethal with mutations affecting the late steps of ergosterol synthesis (erg2 to erg6). Defects in cell polarity and actin organization were observed in the cdc50{Delta} erg3{Delta} mutant. In particular, actin patches, which are normally found at cortical sites, were assembled intracellularly along with their assembly factors, including Las17p, Abp1p, and Sla2p. The exocytic SNARE Snc1p, which is recycled by an endocytic route, was also intracellularly accumulated, and inhibition of endocytic internalization suppressed the cytoplasmic accumulation of both Las17p and Snc1p. Simultaneous loss of both phospholipid asymmetry and sterol structural integrity could lead to accumulation of endocytic intermediates capable of initiating assembly of actin patches in the cytoplasm.


This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–05–0452) on September 29, 2005.

Abbreviations used: 3HA, three tandem repeats of the influenza virus hemagglutinin epitope; 5-FOA, 5-fluoroorotic acid; DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; GC-MS, gas chromatography-mass spectrometry; GFP, green fluorescent protein; LAT-A, latrunculin-A; mRFP1, monomeric red fluorescent protein 1; PS, phosphatidylserine; TGN, trans-Golgi network; TRITC, tetramethylrhodamine B isothiocyanate.

Address correspondence to: Kazuma Tanaka (k-tanaka{at}igm.hokudai.ac.jp).




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