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J. Biol. Chem., Vol. 277, Issue 18, 15486-15498, May 3, 2002
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From the The 26 S proteasome, a complex
between the 20 S proteasome and 19 S regulatory units, catalyzes
ATP-dependent degradation of unfolded and ubiquitinated
proteins in eukaryotes. We have identified previously 20 S and
activated 20 S proteasomes in Trypanosoma brucei, but not
26 S proteasome. However, the presence of 26 S proteasome in T. brucei was suggested by the hydrolysis of casein by cell lysate,
a process that requires ATP but is inhibited by lactacystin, and the
lactacystin-sensitive turnover of ubiquitinated proteins in the intact
cells. T. brucei cDNAs encoding the six proteasome
ATPase homologues (Rpt) were cloned and expressed. Five of the six
T. brucei Rpt cDNAs, except for Rpt2, were capable of
functionally complementing the corresponding rpt deletion
mutants of Saccharomyces cerevisiae. Immunoblots showed the
presence in T. brucei lysate of the Rpt proteins, which
co-fractionated with the yeast 19 S proteasome complex by gel
filtration and localized in the 19 S fraction of a glycerol gradient.
All the Rpt and putative 19 S non-ATPase (Rpn) proteins were
co-immunoprecipitated from T. brucei lysate by individual
anti-Rpt antibodies. Treatment of T. brucei cells with a
chemical cross-linker resulted in co-immunoprecipitation of 20 S
proteasome with all the Rpt and Rpn proteins that sedimented in a
glycerol gradient to the position of 26 S proteasome. These data
demonstrate the presence of 26 S proteasome in T. brucei cells, which apparently dissociate into 19 S and 20 S complexes upon
cell lysis. RNA interference to block selectively the expression of
proteasome 20 S core and Rpt subunits resulted in significant accumulation of ubiquitinated proteins accompanied by cessation of cell
growth. Expression of yeast RPT2 gene in T. brucei Rpt2-deficient cells could not rescue the lethal
phenotype, thus confirming the incompatibility between the two Rpt2s.
The T. brucei 11 S regulator (PA26)-deficient RNA
interference cells grew normally, suggesting the dispensability of
activated 20 S proteasome in T. brucei.
An Easily Dissociated 26 S Proteasome Catalyzes an Essential
Ubiquitin-mediated Protein Degradation Pathway in Trypanosoma
brucei*,
,,,¶,
Department of Pharmaceutical Chemistry and
the § Department of Microbiology and Immunology,
University of California, San Francisco, California 94143-0446
*
This work was supported in part by National Institutes of
Health R01 Grants AI-21786 (to C. C. W.) and GM-45335 (to P. C.).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.
The on-line version of this article (available at
http://www.jbc.org) contains Tables I-III and Figs.
I-VI.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF227499 to AF227504.
¶ Recipient of a United Negro College Fund Merck Science Initiative Fellowship.
To whom correspondence should be addressed. Tel.:
415-476-1321; Fax: 415-476-3382; E-mail: ccwang@cgl.ucsf.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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