Carbon Source-dependent assembly of the Snf1p kinase complex in Candida albicans.
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 beta 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 (iTraqtrade mark) 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.
Corvey
Carsten
C
Institute for Microbiology, Johann Wolfgang Goethe-University of Frankfurt, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany. corvey@iachem.uni-frankfurt.de
Koetter
Peter
P
Beckhaus
Tobias
T
Hack
Jeremy
J
Hofmann
Sandra
S
Hampel
Martin
M
Stein
Torsten
T
Karas
Michael
M
Entian
Karl-Dieter
KD
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics*; D002244:Carbon / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010957:Plasmids; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D002244:Carbon; C071570:SNF1-related protein kinases; D017346:Protein Serine-Threonine Kinases; C078651:SNF4 protein, S cerevisiae; D055372:AMP-Activated Protein Kinases