| pmid | 10515935 | |||||
|---|---|---|---|---|---|---|
| title | Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae. | |||||
| authors |
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| journal | J Bacteriol | |||||
| year | 1999 | |||||
| full_text_available | true | |||||
| full_text_extraction_method | html | |||||
| pmcid | PMC103780 | |||||
| doi | 10.1128/JB.181.20.6441-6448.1999 |
Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.
Authors: Athenstaedt K, Zweytick D, Jandrositz A, Kohlwein SD, Daum G Journal: J Bacteriol (1999) DOI: 10.1128/JB.181.20.6441-6448.1999 PMC: PMC103780
- J Bacteriol. 1999 Oct;181(20):6441-8. doi: 10.1128/JB.181.20.6441-6448.1999.
Identification and characterization of major lipid particle proteins of the yeast Saccharomyces cerevisiae.
Athenstaedt K(1), Zweytick D, Jandrositz A, Kohlwein SD, Daum G.
Author information: (1)Institut für Biochemie und Lebensmittelchemie, Technische Universität and SFB Biomembrane Research Center, Petersgasse 12/2, A-8010 Graz, Austria.
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.
DOI: 10.1128/JB.181.20.6441-6448.1999 PMCID: PMC103780 PMID: 10515935 [Indexed for MEDLINE]
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.
RESULTS
To identify proteins of yeast lipid particles, polypeptides of a highly enriched lipid particle fraction were separated by SDS-PAGE (Fig. 2 ), reisolated from the gel, and subjected to mass spectrometric analysis as described in Materials and Methods. This strategy led to the identification of the major yeast lipid particle proteins as summarized in Table 1 . In some of the bands excised from the gel (Fig. 2 ), more than one protein was detected, namely Fat1p and Faa4p (the amount of Faa4p was greater than Fat1p), Erg7p and Fat1p (the amount of Erg7p was greater than Fat1p), Tgl1p and the YOR059c gene product (approximately equal amounts), Erg6p and the YDL193w gene product (equal amounts), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Yju3p (the amount of Yju3p was greater than GAPDH). Since the mass spectrometric method employed did not allow exact quantification of proteins identified in the same band, the molar ratio could be only roughly estimated. Some proteins or their polypeptide fragments were identified in several bands, namely Fat1p, Tgl1p, Erg6p, and the gene product of YIL124w (Fig. 2 ). The reason for this finding is most likely degradation of proteins during the isolation procedure. Alternatively, different posttranslationally modified forms of proteins might be present on the lipid particle surface, although such modifications could not be demonstrated by the methods used for this study.
DISCUSSION
Mass spectrometric analysis was used to identify major proteins of lipid particles of the yeast S. cerevisiae . This approach supported previous findings concerning the localization of Erg6p and Erg1p to lipid particles ( 27 , 28 ). In addition, several proteins with known function could be attributed to the lipid particle fraction during this study, namely Erg7p, Faa1p, Faa4p, and Fat1p ( 4 , 10 , 22 , 23 , 40 ). Furthermore, some novel gene products encoded by unassigned ORFs were identified as lipid particle components.
What is the physiological role of lipid particle proteins? Since several enzymes of ergosterol synthesis are located on lipid particles, it is tempting to speculate that these proteins actively participate in cellular sterol formation. This is very likely for Erg6p ( 27 ) and Erg7p ( 4a ), which are enzymatically active in lipid particle preparations. Similarly, a glycerol-3-phosphate acyltransferase encoded by the unidentified GAT gene and the 1-acylglycerol-3-phosphate acyltransferase Slc1p, which contribute to phosphatidic acid biosynthesis, were previously identified as lipid particle components ( 3 , 7 , 43 ). It has to be mentioned, however, that Slc1p escaped detection by the mass spectrometric approach most likely due to its low abundance. None of the unassigned ORFs could be identified as GAT since lipid particles of all deletion strains tested contained wild-type levels of glycerol-3-phosphate acyltransferase activity ( 2a ).
In contrast to the above-mentioned enzymes, it was shown that squalene epoxidase (Erg1p) of isolated lipid particles is not enzymatically active in vitro, whereas Erg1p present in the endoplasmic reticulum fraction exhibits enzymatic activity ( 28 ). It was argued that a component, probably a reductase, that is present in the endoplasmic reticulum but absent from lipid particles may be the missing cofactor. Interaction of the endoplasmic reticulum with lipid particles may activate Erg1p of the latter compartment.
The presence of enzymatically inactive proteins on the surface of lipid particles, as described for Erg1p, may also be interpreted as a regulatory phenomenon. If Erg1p of lipid particles does not contribute to ergosterol synthesis in vivo, this protein might be put on hold on the surface of this compartment for a situation which requires enhancement of lipid biosynthesis. Under these conditions, enzymes could be immediately mobilized from lipid particles and translocated to their site of activation, e.g., the endoplasmic reticulum, thus providing lipids within a short time without new polypeptide synthesis. The idea of depositing proteins in lipid particles during formation of this compartment has been previously advocated by Lum and Wright ( 31 ) when studying overexpression of 3-hydroxy-3-methylglutaryl CoA reductase in Schizosaccharomyces pombe . This enzyme, which accumulated first in so-called karmellae, was deposited in lipid particles upon degradation of the former organelle.
The function of several gene products located on yeast lipid particles remains to be demonstrated. With one exception, YDL193w, a deletion of ORFs encoding lipid particle proteins affected neither cell viability nor the formation of lipid particles in a significant way. All these deletion strains contain lipid particles of normal size and physical properties as shown by microscopic inspection and isolation of the respective fractions ( 44 ). We can only speculate at present that some proteins located on the surface of lipid particles may be involved in the deposition of triacylglycerols and/or steryl esters in or mobilization of these lipids from this compartment. The gene product of YMR313c may be a candidate for such a function because the strain having a deletion of this ORF accumulates triacylglycerols to some extent (Table 4 ).
An intriguing question concerns targeting and transfer of proteins to lipid particles. Through localization studies of Erg1p ( 28 ) and of Slc1p and Gat1p ( 3 ), it was demonstrated that lipid particles and the endoplasmic reticulum share a certain set of proteins and thus appear to be related compartments. At present, we can only speculate about the relationship of these two subcellular fractions. The fact that (i) all lipid particle proteins characterized so far are involved in lipid metabolism and (ii) most lipid particle proteins contain either none or only a small number of TM domains may serve as the basis for the following hypothesis (Fig. 3 A). Several enzymes involved in lipid synthesis may be located in specific domains of the endoplasmic reticulum. Clustering of these enzymes might cause local accumulation of newly formed lipids, especially those which are unable to integrate into a phospholipid bilayer, namely triacylglycerols and steryl esters. These neutral lipids may form microdroplets (preforms of lipid particles) between the two leaflets of the endoplasmic reticulum membrane bilayer which bud off after reaching a certain size. The presence of steryl ester synthases Are1p and Are2p in the endoplasmic reticulum ( 45 ) is in line with this model. Recent findings from our laboratory ( 3a ) suggest that the endoplasmic reticulum is also the major site of triacylglycerol synthesis, indicating that both neutral lipid species of lipid particles are formed in the same compartment. It has to be noted, however, that triacylglycerol synthase activity has previously been attributed to the lipid particle fraction by Christiansen ( 7 ). The assay used by this author, however, did not allow for distinguishing between acylation of the substrate diacylglycerol and transacylation reactions.
Fluorescence microscopic evidence obtained recently in our laboratory ( 23a ) demonstrated the appearance of small, newly formed lipid particles in proximity to the endoplasmic reticulum, thus supporting the model presented in Fig. 3 A. According to the molecular shape concept ( 18 ), PtdIns-rich domains in the endoplasmic reticulum might facilitate the budding process. This hypothesis is in line with the finding that PtdIns comprises approximately 30% of total lipid particle phospholipids ( 27 ), whereas PtdIns is only a minor component among endoplasmic reticulum bulk phospholipids. During the budding process, newly formed lipid particles may be enwrapped by an endoplasmic reticulum-derived phospholipid monolayer, which indeed forms the surface membrane of lipid particles ( 27 ). Proteins with none or only a low number of TM domains initially present in the endoplasmic reticulum may remain associated with the phospholipid monolayer of lipid particles during the budding process, whereas proteins with typical TM regions may be largely excluded.
Although the above-mentioned hypothesis of lipid particle biosynthesis is consistent with experimental evidence obtained during our studies and compatible with the theory of oil body formation in plants ( 16 ), alternative possibilities of lipid particle formation should be considered. As an example, proteins could be directed to the surface of preformed lipid particles by a targeting signal (Fig. 3 B). Although no such typical motifs were found in lipid particle proteins, signals based on conformational properties that escaped our attention may be important in that respect. As a further possible mechanism for assembly of proteins to lipid particles, transport of proteins through vesicle flux might be considered (Fig. 3 C). Experimental evidence for such a mechanism, however, is also missing. The protein encoded by YBR177c, which is slightly homologous to a probable human membrane receptor (HPS1) (Table 1 ), might be regarded as a candidate for facilitating such a vesicle docking process. The fact that the mutant with a deletion of YBR177c contains lipid particles with a slightly different protein pattern than the wild type could be an argument for this hypothesis. A detailed analysis of this mutant and characterization of the YBR177c gene product will be required to address this question.