Mannosyl-Diinositolphospho-Ceramide, the Major Yeast Plasma Membrane Sphingolipid, Governs Toxicity of Kluyveromyces lactis Zymocin

doi:10.1128/EC.4.5.879-889.2005

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Mannosyl-Diinositolphospho-Ceramide, the Major Yeast Plasma Membrane Sphingolipid, Governs Toxicity of Kluyveromyces lactis Zymocin

Sabrina Zink,¹^, Constance Mehlgarten,¹^, Hiroko K. Kitamoto,²^, Junko Nagase,² Daniel Jablonowski,¹ Robert C. Dickson,³ Michael J. R. Stark,⁴ and Raffael Schaffrath¹^*

Biologicum, Institut für Genetik, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany,¹ National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan,² Department of Molecular and Cellular Biochemistry, University of Kentucky, College of Medicine, 800 Rose Street, Lexington, Kentucky,³ Division of Gene Regulation and Expression, School of Life Sciences, University of Dundee, MSI/WTB Complex, Dow Street, Dundee DD1 5EH, United Kingdom⁴

Received 25 January 2005/ Accepted 7 March 2005

Kluyveromyces lactis zymocin, a trimeric ( ${alpha}$ ß ${gamma}$ ) proteintoxin complex, inhibits proliferation of Saccharomyces cerevisiaecells. Here we present an analysis of kti6 mutants, which resistexogenous zymocin but are sensitive to intracellular expressionof its inhibitory ${gamma}$ -toxin subunit, suggesting that KTI6 encodesa factor needed for toxin entry into the cell. Consistent withaltered cell surface properties, kti6 cells resist hygromycinB, syringomycin E, and nystatin, antibiotics that require intactmembrane potentials or provoke membrane disruption. KTI6 isallelic to IPT1, coding for mannosyl-diinositolphospho-ceramide[M(IP)₂C] synthase, which produces M(IP)₂C, the major plasmamembrane sphingolipid. kti6 membranes lack M(IP)₂C and sphingolipidmutants that have reduced levels of M(IP)₂C precursors, includingthe sphingolipid building block ceramide survive zymocin. Inaddition, kti6/ipt1 cells allow zymocin docking but preventimport of its toxic ${gamma}$ -subunit. Genetic analysis indicates thatKti6 is likely to act upstream of lipid raft proton pump Kti10/Pma1,a previously identified zymocin sensitivity factor. In sum,M(IP)₂C operates in a plasma membrane step that follows recognitionof cell wall chitin by zymocin but precedes the involvementof elongator, the potential toxin target.

* Corresponding author. Mailing address: Biologicum, Institut für Genetik, Martin-Luther-Universität Halle-Wittenberg, Weinbergweg 10, D-06120 Halle (Saale), Germany. Phone: 49-345-5526333. Fax: 49-345-5527151. E-mail: schaffrath{at}genetik.uni-halle.de.

These authors contributed equally to the work.

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