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J. Biol. Chem., Vol. 278, Issue 9, 6687-6695, February 28, 2003
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From the Centro de Biología Molecular "Severo Ochoa,"
CSIC and Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain and § Department of Cell Biology,
Harvard Medical School, Boston, Massachusetts 02115
We report the functional characterization
of RPN6, an essential gene from Saccharomyces
cerevisiae encoding the proteasomal subunit Rpn6p. For this
purpose, conditional mutants that are able to grow on galactose but not
on glucose were obtained. When these mutants are shifted to glucose,
Rpn6p depletion induces several specific phenotypes. First,
multiubiquitinated proteins accumulate, indicating a defect in
proteasome-mediated proteolysis. Second, mutant yeasts are arrested as
large budded cells with a single nucleus and a 2C DNA content;
in addition, the spindle pole body is duplicated, indicating a general
cell cycle defect related to the turnover of G2-cyclins
after DNA synthesis. Clb2p and Pds1p, but not Sic1p, accumulate in the
arrested cells. Depletion of Rpn6p affects both the structure and the
peptidase activity of proteasomes in the cell. These results implicate
Rpn6p function in the specific recognition of a subset of substrates
and point to a role in maintaining the correct quaternary structure of
the 26 S proteasome.
Rpn6p, a Proteasome Subunit from Saccharomyces
cerevisiae, Is Essential for the Assembly and Activity of the 26 S Proteasome*
,
*
This work was supported by Grant PM1999-0108 from the
Ministerio de Ciencia y Tecnología (Spain) and National
Institutes of Health Grant GM43601 (to D. F.) and by an institutional
grant to the Centro de Biología Molecular "Severo Ochoa"
from Fundación Ramón Areces (Madrid).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Recipient of a Formación Personal Investigador
fellowship from the Comunidad Autónoma de Madrid.
¶
To whom correspondence should be addressed. Tel.:
34-91-397-8676; Fax: 34-91-397-4799; E-mail:
miguel.remacha@uam.es.
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