In Saccharomyces cerevisiae, the ubiquitin-like protein Rub1p (related to ubiquitin 1 protein) covalently attaches to the cullin protein Cdc53p (cell division cycle 53 protein), a subunit of a class of ubiquitin E3 ligases named SCF (Skp1–Cdc53–F-box protein) complex. We identified Rtt101p (regulator of Ty transposition 101 protein, where Ty stands for transposon of yeast), initially found during a screen for proteins to confer retrotransposition suppression, and Cul3p (cullin 3 protein), a protein encoded by the previously uncharacterized open reading frame YGR003w, as two new in vivo targets for Rub1p conjugation. These proteins show significant identity with Cdc53p and, therefore, are cullin proteins. Modification of Cul3p is eliminated by deletion of the Rub1p pathway through disruption of either RUB1 or its activating enzyme ENR2/ULA1. The same disruptions in the Rub pathway decreased the percentage of total Rtt101p that is modified from approx. 60 to 30%. This suggests that Rtt101p has an additional RUB1- and ENR2-independent modification. All modified forms of Rtt101p and Cul3p were lost when a single lysine residue in a conserved region near the C-terminus was replaced by an arginine residue. These results suggest that this lysine residue is the site of Rub1p-dependent and -independent modifications in Rtt101p and of Rub1p-dependent modification in Cul3p. An rtt101Δ strain was hypersensitive to thiabendazole, isopropyl (N-3-chlorophenyl) carbamate and methyl methanesulphonate, but rub1Δ strains were not. Whereas rtt101Δ strains exhibited a 14-fold increase in Ty1 transposition, isogenic rub1Δ strains did not show statistically significant increases. Rtt101K791Rp, which cannot be modified, complemented for Rtt101p function in a transposition assay. Altogether, these results suggest that neither the RUB1-dependent nor the RUB1-independent form of Rtt101p is required for Rtt101p function. The identification of additional Rub1p targets in S. cerevisiae suggests an expanded role for Rub in this organism.
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January 2004
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Research Article|
January 15 2004
Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo
Jose M. LAPLAZA;
*Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
†Genetics Graduate Group, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
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Magnolia BOSTICK;
Magnolia BOSTICK
2
*Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
‡Biochemistry and Molecular Biology Graduate Group, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
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Derek T. SCHOLES;
Derek T. SCHOLES
§Molecular Genetics Program, Wadsworth Center, P.O. Box 22002, Albany, NY 12201-2002, U.S.A.
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M. Joan CURCIO;
M. Joan CURCIO
§Molecular Genetics Program, Wadsworth Center, P.O. Box 22002, Albany, NY 12201-2002, U.S.A.
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Judy CALLIS
Judy CALLIS
3
*Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
†Genetics Graduate Group, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
‡Biochemistry and Molecular Biology Graduate Group, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, U.S.A.
3To whom correspondence should be addressed (e-mail jcallis@ucdavis.edu).
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Publisher: Portland Press Ltd
Received:
May 22 2003
Revision Received:
September 24 2003
Accepted:
October 01 2003
Accepted Manuscript online:
October 01 2003
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2004
2004
Biochem J (2004) 377 (2): 459–467.
Article history
Received:
May 22 2003
Revision Received:
September 24 2003
Accepted:
October 01 2003
Accepted Manuscript online:
October 01 2003
Citation
Jose M. LAPLAZA, Magnolia BOSTICK, Derek T. SCHOLES, M. Joan CURCIO, Judy CALLIS; Saccharomyces cerevisiae ubiquitin-like protein Rub1 conjugates to cullin proteins Rtt101 and Cul3 in vivo. Biochem J 15 January 2004; 377 (2): 459–467. doi: https://doi.org/10.1042/bj20030755
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