Carbon source-dependent assembly of the Snf1p kinase complex in Candida albicans
Carbon source-dependent assembly of the Snf1p kinase complex in Candida albicans
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DOI：
10.1074/jbc.m503719200
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发表时间：
2005-07-08
影响因子：
4.8
通讯作者：
Entian, KD
中科院分区：
文献类型：
--
作者：
Corvey, C;Koetter, P;Entian, KD
Snf1p/ amp活化激酶参与应激反应的转录、代谢和发育调节。在酿酒酵母中，Snf1p （Cat1p）是碳水化合物代谢的关键调节因子之一，而cat1 （snf1）突变体在不可发酵的碳源下无法生长。在白色念珠菌中，Snf1p是一种必需蛋白，即使以葡萄糖为碳源，细胞也依赖于功能性Snf1激酶。我们对CaSnf1p配合物进行了串联亲和纯化和质谱分析，发现配合物的组成随碳源的变化而变化。鉴定出三个亚基，其中一个被命名为CaSnf4p，因为它与ScSnf4蛋白同源，并且各自的CaSNF4基因可以补充酿酒葡萄球菌snf4突变体。另外两个蛋白与酿酒酵母激酶β亚基ScGal83p、ScSip2p和ScSip1p相似。这两个基因在酿酒酵母gal83，sip1，sip2三重缺失突变体中都具有支架功能，并根据其支架功能命名为CaKIS1p和CaKIS2p。基质辅助激光解吸电离肽质量指纹图谱分析表明，与以葡萄糖为碳源生长的细胞相比，CaKis2p在Snf1p复合物中的掺入量减少。为了验证不同的复合物组合，采用了稳定同位素标记技术（iTraq (TM)），证实乙醇使CaKis2p减少了3倍。酵母双杂交分析证实了相互作用伙伴，这些结果表明CaKis2蛋白的激活域在酿酒酵母支架亚基中尚未报道。
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 (iTraq (TM)) 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.