Carbon Source-dependent Assembly of the Snf1p Kinase Complex inCandidaalbicans

摘要

The Snf1p/AMP-activated kinases are involved in transcriptional, metabolic,and developmental regulation in response to stress. In Saccharomycescerevisiae, Snf1p (Cat1p) is one of the key regulators of carbohydratemetabolism, and cat1 (snf1) mutants fail to grow withnon-fermentable carbon sources. In Candida albicans, Snf1p is anessential protein and cells depend on a functional Snf1 kinase even withglucose as carbon source. We investigated the CaSnf1p complex aftertandem affinity purification and mass spectrometric analysis and show that thecomplex composition changes with the carbon source provided. Three subunitswere identified, one of which was named CaSnf4p because of itshomology to the ScSnf4 protein and the respective CaSNF4gene could complement a S. cerevisiae snf4 mutant. The other twoproteins revealed similarities to the S. cerevisiae kinase βsubunits ScGal83p, ScSip2p, and ScSip1p. Both genescomplemented the scaffold function in a S. cerevisiae gal83,sip1,sip2triple deletion mutant and were named according to their scaffold function asCaKIS1p and CaKIS2p. Matrix-assisted laser desorptionionization peptide mass fingerprint analysis indicated that CaKis2pis N-terminal myristoylated and the incorporation of CaKis2p in theSnf1p complex was reduced when compared with cells grown with glucose as acarbon source. To verify the different complex assemblies, a stable isotopelabeling technique (iTraq™) was employed, confirming a 3-fold decreaseof CaKis2p with ethanol. Yeast two-hybrid analysis confirmed theinteraction partners, and these results showed an activator domain for theCaKis2 protein that has not been reported for S. cerevisiaescaffold subunits. The Snf1p/AMP-activated kinases are involved in transcriptional, metabolic,and developmental regulation in response to stress. In Saccharomycescerevisiae, Snf1p (Cat1p) is one of the key regulators of carbohydratemetabolism, and cat1 (snf1) mutants fail to grow withnon-fermentable carbon sources. In Candida albicans, Snf1p is anessential protein and cells depend on a functional Snf1 kinase even withglucose as carbon source. We investigated the CaSnf1p complex aftertandem affinity purification and mass spectrometric analysis and show that thecomplex composition changes with the carbon source provided. Three subunitswere identified, one of which was named CaSnf4p because of itshomology to the ScSnf4 protein and the respective CaSNF4gene could complement a S. cerevisiae snf4 mutant. The other twoproteins revealed similarities to the S. cerevisiae kinase βsubunits ScGal83p, ScSip2p, and ScSip1p. Both genescomplemented the scaffold function in a S. cerevisiae gal83,sip1,sip2triple deletion mutant and were named according to their scaffold function asCaKIS1p and CaKIS2p. Matrix-assisted laser desorptionionization peptide mass fingerprint analysis indicated that CaKis2pis N-terminal myristoylated and the incorporation of CaKis2p in theSnf1p complex was reduced when compared with cells grown with glucose as acarbon source. To verify the different complex assemblies, a stable isotopelabeling technique (iTraq™) was employed, confirming a 3-fold decreaseof CaKis2p with ethanol. Yeast two-hybrid analysis confirmed theinteraction partners, and these results showed an activator domain for theCaKis2 protein that has not been reported for S. cerevisiaescaffold subunits. Candida albicans is a widely distributed commensal fungus that iscarried as a part of the human microbial flora. However, it is also anopportunistic pathogen that can cause serious infections, particularly inimmunocompromised individuals(1Odds F. Candida andCandidiosis. 2nd Ed. Leicester University Press, Leicester, UK1988: 279-313Google Scholar). In C. albicans,Snf1p (CaSnf1p, NCBI accession number 46437276) seems to be essentialfor growth; moreover, CaSnf1p has the ability to complement thesnf1 mutant of Saccharomyces cerevisiae(2Petter R. Chang Y.C. Kwon-Chung K.J. Infect. Immun. 1997; 65: 4909-4917Crossref PubMed Google Sc