Analysis of Opi1p repressor mutants
- 138 Downloads
Opi1p is the only known repressor protein specific to the phospholipid biosynthetic pathway. Opi1p is required for repression in response to inositol and choline supplementation. However, the mechanism of Opi1p repression is not completely understood. In part, this is because previously identified opi1 mutants contained nonsense mutations and thus provided little insight into the mechanism of Opi1p function. We have recently reported isolating novel opi1 mutants (rum and dim mutants) that contain missense mutations. Here, we show that these opi1 mutants produce Opi1p product at levels comparable to a wild-type strain. However, these mutants mis-regulate expression of two target genes, INO2-HIS3 and INO1-lacZ, and are also defective in autoregulation. An opi1-S339F mutant is particularly interesting because it completely eliminated autoregulation, but only abated regulation of an INO1-lacZ reporter. Two of the mutations in OPI1 (V343Q and S339F) provide genetic evidence for an interaction between Opi1p and the Ino2p activator since they reside in a region of Opi1p recently shown to interact with Ino2p in vitro. A third mutation (L252F) resides in a region of Opi1p with no known function.
KeywordsSaccharomyces cerevisiae Repressor Phospholipid biosynthesis Regulation Autoregulation INO1 INO2
The authors thank Kyle Gardenour, Mary Beth Gardocki, Kaidan Su, and Meng Chen for helpful discussions and assistance with editing. This work was supported by a National Science Foundation grant (MCB-0110408) to JML.
- Cok SJ, Martin CG, Gordon JI (1998) Transcription of INO2 and INO4 is regulated by the state of protein N-myristoylation in Saccharomyces cerevisiae. Nucl Acids Res 23:1426–1433Google Scholar
- Gardenour KR, Levy J, Lopes JM (2004) Identification of novel dominant INO2c mutants with an Opi-phenotype. Mol Micro (in press)Google Scholar
- Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415:141–147PubMedCrossRefGoogle Scholar
- Greenberg ML, Lopes JM (1996) Genetic regulation of phospholipid biosynthesis in Saccharomyces cerevisiae. Micro Rev 60:1–20Google Scholar
- Henry SA, Patton-Vogt JL (1998) Genetic regulation of phospholipid metabolism: yeast as a model eukaryote. Prog Nucl Acid Res Mol Biol 61:133–179Google Scholar
- Sherman F (1991) Getting started with yeast. Meth Enzymol 94:1–20Google Scholar