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J. Biol. Chem., Vol. 280, Issue 27, 25323-25330, July 8, 2005
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¶
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From the
Institutes for ||Microbiology and
Pharmaceutical Chemistry, Johann Wolfgang
Goethe-University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt,
Germany
The Snf1p/AMP-activated kinases are involved in transcriptional, metabolic,
and developmental regulation in response to stress. In Saccharomyces
cerevisiae, Snf1p (Cat1p) is one of the key regulators of carbohydrate
metabolism, and cat1 (snf1) mutants fail to grow with
non-fermentable carbon sources. In Candida albicans, Snf1p is an
essential protein and cells depend on a functional Snf1 kinase even with
glucose as carbon source. We investigated the CaSnf1p complex after
tandem affinity purification and mass spectrometric analysis and show that the
complex composition changes with the carbon source provided. Three subunits
were identified, one of which was named CaSnf4p because of its
homology to the ScSnf4 protein and the respective CaSNF4
gene could complement a S. cerevisiae snf4 mutant. The other two
proteins revealed similarities to the S. cerevisiae kinase
subunits ScGal83p, ScSip2p, and ScSip1p. Both genes
complemented the scaffold function in a S. cerevisiae gal83,sip1,sip2
triple deletion mutant and were named according to their scaffold function as
CaKIS1p and CaKIS2p. Matrix-assisted laser desorption
ionization peptide mass fingerprint analysis indicated that CaKis2p
is N-terminal myristoylated and the incorporation of CaKis2p in the
Snf1p complex was reduced when compared with cells grown with glucose as a
carbon source. To verify the different complex assemblies, a stable isotope
labeling technique (iTraqTM) was employed, confirming a 3-fold decrease
of CaKis2p with ethanol. Yeast two-hybrid analysis confirmed the
interaction partners, and these results showed an activator domain for the
CaKis2 protein that has not been reported for S. cerevisiae
scaffold subunits.
Received for publication, April 5, 2005 , and in revised form, May 4, 2005.
* The costs of publication of this article were defrayed in part by the payment of page charges. This 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 two supplemental tables.
Both authors contributed equally to this work.
¶ To whom correspondence may be addressed. Tel.: 49-69-798-29925; Fax: 49-69-798-29918; E-mail: corvey{at}iachem.uni-frankfurt.de. ** To whom correspondence may be addressed. Tel.: 49-69-798-29529; Fax: 49-69-798-29527; E-mail: koetter{at}em.uni-frankfurt.de.
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