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J. Biol. Chem., Vol. 281, Issue 37, 27158-27166, September 15, 2006

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Bem1p Is a Positive Regulator of the Homotypic Fusion of Yeast Vacuoles^*

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844

Docked vacuoles are believed to undergo rapid lipid mixing during hemifusion and then a slow, rate-limiting completion of fusion and mixing of lumenal contents. Previous genomic analysis has suggested that Bem1p, a scaffold protein critical for cell polarity, may support vacuole fusion. We now report that bem1 strains have fragmented vacuoles (vps class B and C). During in vitro fusion reactions, vacuoles from bem1 strains showed a strong reduction in the rate of lipid mixing when compared with vacuoles from the BEM1 parent. The reduction in the overall rate of fusion with bem1 vacuoles was modest, consistent with lipid mixing as a non-rate-limiting step in the pathway. Although the fusion of either BEM1 (wild-type) or bem1 vacuoles is stimulated by recombinant Bem1p, the lipid mixing of docked bem1 vacuoles is highly dependent on rBem1p under certain reaction conditions. Bem1p-stimulated lipid mixing is blocked by well characterized fusion inhibitors including lipid ligands and antibodies to Ypt7p, Vps33p, and Vam3p. Although full-length Bem1p is required for maximal stimulation, a truncation mutant comprising the SH3 domains and the Phox homology (PX) domain retains modest stimulatory activity. In contrast to an earlier report (Han, B. K., Bogomolnaya, L. M., Totten, J. M., Blank, H. M., Dangott, L. J., and Polymenis, M. (2005) Genes Dev. 19, 2606–2618), we did not find phosphorylation of Bem1p at Ser-72 to be required for Bem1p-stimulated fusion. Taken together, Bem1p is a positive regulator of lipid mixing during vacuole hemifusion and fusion.

Received for publication, June 12, 2006 , and in revised form, July 18, 2006.

^* This work was supported by a grant from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, Dartmouth Medical School, 7200 Vail Bldg., Hanover, NH 03755-3844. Tel.: 603-650-1701; Fax: 603-650-1353; E-mail: Bill.Wickner{at}Dartmouth.edu.

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