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Originally published In Press as doi:10.1074/mcp.M400123-MCP200 on February 16, 2005.
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Molecular & Cellular Proteomics 4:662-672, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy*,S

Klaus Natter{ddagger}, Peter Leitner{ddagger}, Alexander Faschinger{ddagger}, Heimo Wolinski{ddagger}, Stephen McCraith§,¶, Stanley Fields§,||,** and Sepp D. Kohlwein{ddagger},{ddagger}{ddagger}

From the {ddagger} Institute of Molecular Biosciences, Spezialforschungsbereich Biomembrane Research Center, University of Graz, Schubertstr. 1, A8010 Graz, Austria and the § Departments of Genome Sciences and Medicine and the || Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins (~9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

{ddagger}{ddagger} To whom correspondence should be addressed. Tel.: 43-316-380-5487; Fax: 43-316-380-9857; E-mail: sepp.kohlwein{at}uni-graz.at


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