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J. Biol. Chem., Vol. 280, Issue 6, 4270-4278, February 11, 2005

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Characterizing the Sphingolipid Signaling Pathway That Remediates Defects Associated with Loss of the Yeast Amphiphysin-like Orthologs, Rvs161p and Rvs167p^*

Melody Germann, Evelyn Swain, Lawrence Bergman, and Joseph T. Nickels, Jr.¶

From the Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267–277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.

Received for publication, November 3, 2004 , and in revised form, November 18, 2004.

^* This work was supported by National Institutes of Health Grant HL67401 (to J. T. N.) and Basil O'Connor Starter Scholarship Award FY99-277 (to J. T. N.). 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.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N. 15th St., NCB 11115, MS 497, Philadelphia, PA 19102. Tel.: 215-762-1941; Fax: 215-762-4452; E-mail: JN27{at}drexel.edu.

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