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Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p
Journal article   Open access   Peer reviewed

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
The Journal of biological chemistry, v 280(6), pp 4270-4278
11 Feb 2005
DOI: https://doi.org/10.1074/jbc.M412454200
PMID: 15561700
url
https://doi.org/10.1074/jbc.M412454200View
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Abstract

Sphingolipids - chemistry Temperature Immunoprecipitation Actins - metabolism Saccharomyces cerevisiae - metabolism Lipids - chemistry Nerve Tissue Proteins - chemistry Glucan Endo-1,3-beta-D-Glucosidase - chemistry Gene Deletion beta-Galactosidase - metabolism Actins - chemistry Acetyltransferases Protein Structure, Tertiary Signal Transduction Membrane Microdomains - chemistry Sphingolipids - metabolism Meiosis Cytoskeletal Proteins - chemistry Plasmids - metabolism Blotting, Western Proline - chemistry Two-Hybrid System Techniques Phenotype Microfilament Proteins Alleles Saccharomyces cerevisiae Proteins - metabolism Cytoskeleton - metabolism Protein Binding Epitopes - chemistry Mutation Subcellular Fractions Microscopy, Fluorescence Lac Operon Saccharomyces cerevisiae Proteins - chemistry
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.

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Biochemistry & Molecular Biology
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