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Enhanced levels of Pis1p (phosphatidylinositol synthase) improve the growth of Saccharomyces cerevisiae cells deficient in Rsp5 ubiquitin ligase.

ABSTRACT: The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.

SUBMITTER: Kaliszewski P

PROVIDER: S-EPMC1409703 | biostudies-other | 2006 Apr

REPOSITORIES: biostudies-other

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Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae.

Project description:We characterized a temperature-sensitive mutant of Saccharomyces cerevisiae in which a mini-chromosome was unstable at a high temperature and cloned a new gene which encodes a basic and hydrophilic protein (110 kDa). The disruption of this gene caused the same temperature-sensitive growth as the original mutation. By using the two-hybrid system, we further isolated RSP5 (reverses Spt- phenotype), which encodes a hect (homologous to E6-AP C terminus) domain, as a gene encoding a ubiquitin ligase. Thus, we named our gene BUL1 (for a protein that binds to the ubiquitin ligase). BUL1 seems to be involved in the ubiquitination pathway, since a high dose of UBI1, encoding a ubiquitin, partially suppressed the temperature sensitivity of the bul1 disruptant as well as that of a rsp5 mutant. Coexpression of RSP5 and BUL1 on a multicopy plasmid was toxic for mitotic growth of the wild-type cells. Pulse-chase experiments revealed that Bul1 in the wild-type cells remained stable, while the bands of Bul1 in the rsp5 cells were hardly detected. Since the steady-state levels of the protein were the same in the two strains as determined by immunoblotting analysis, Bul1 might be easily degraded during immunoprecipitation in the absence of intact Rsp5. Furthermore, both Bul1 and Rsp5 appeared to be associated with large complexes which were separated through a sucrose gradient centrifugation, and Rsp5 was coimmunoprecipitated with Bul1. We discuss the possibility that Bul1 functions together with Rsp5 in protein ubiquitination.

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Rsp5 ubiquitin-protein ligase mediates DNA damage-induced degradation of the large subunit of RNA polymerase II in Saccharomyces cerevisiae.

Project description:Rsp5 is an E3 ubiquitin-protein ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 proteins. We have previously shown that Rsp5 binds and ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 protein closely related to Rsp5, Rpf1/hNedd4, also binds and ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.

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Interactions between Yeast Proteins and Rsp5 Ubiquitin Ligase

2008-12-30 | GSE14218 | GEO

Interactions between Yeast Proteins and Rsp5 Ubiquitin Ligase

Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.

2008-12-30 | E-GEOD-14218 | biostudies-arrayexpress

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase.

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