Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae

Abstract

Lipid particles of the yeast Saccharomyces cerevisiae were isolated at high purity, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Major lipid particle proteins were identified by mass spectrometric analysis, and the corresponding open reading frames (ORFs) were deduced. In silicio analysis revealed that all lipid particle proteins contain several hydrophobic domains but none or only few (hypothetical) transmembrane spanning regions. All lipid particle proteins identified by function so far, such as Erg1p, Erg6p, and Erg7p (ergosterol biosynthesis) and Faa1p, Faa4p, and Fat1p (fatty acid metabolism), are involved in lipid metabolism. Based on sequence homology, another group of three lipid particle proteins may be involved in lipid degradation. To examine whether lipid particle proteins of unknown function are also involved in lipid synthesis, mutants with deletions of the respective ORFs were constructed and subjected to systematic lipid analysis. Deletion of YDL193w resulted in a lethal phenotype which could not be suppressed by supplementation with ergosterol or fatty acids. Other deletion mutants were viable under standard conditions. Strains with YBR177c, YMR313c, and YKL140w deleted exhibited phospholipid and/or neutral lipid patterns that were different from the wild-type strain and thus may be further candidate ORFs involved in yeast lipid metabolism.

FIG. 1

Deletion of eight ORFs encoding yeast lipid particle proteins. Primers were used for the deletion of ORFs by the short flanking homology method. The underlined sequences are homologous to the kanMX4 gene. S1, sequence 1; S2, sequence 2.

FIG. 2

Protein pattern of lipid particles. Lipid particle proteins (LP) were separated by SDS-PAGE, reisolated from the gel, and subjected to mass spectrometric analysis as described in Materials and Methods. ST, standard proteins. The numbering of the bands is the same as that shown in Tables 1 and 2.

FIG. 3

Hypothetical models describing targeting and transfer of proteins to lipid particles. (A) Budding of a neutral lipid-rich domain from the endoplasmic reticulum is accompanied by association of proteins with the phospholipid monolayer of newly formed lipid particles. According to this model, polypeptides without TM regions would preferentially associate with lipid particles. Nasc-LP, nascent lipid particles. (B) Association of proteins with preexisting lipid particles though a targeting signal on the polypeptide. (C) Transport of proteins to preexisting lipid particles by vesicle (V) flow. LP, lipid particles; ER, endoplasmic reticulum.