Purification and characterization of Snf1 kinase complexes containing a defined β subunit composition
Purification and characterization of Snf1 kinase complexes containing a defined β subunit composition
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DOI：
10.1074/jbc.m207058200
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发表时间：
2002-12-27
影响因子：
4.8
通讯作者：
Schmidt, MC
中科院分区：
文献类型：
--
作者：
Nath, N;McCartney, RR;Schmidt, MC
酿酒酵母的Snf 1激酶复合物包含由SIP 1、SIP 2或GAL 83编码的三种可能的β亚基之一。使用Snf 1蛋白C末端上的串联亲和纯化标签，从仅表达三种β亚基之一的细胞中纯化Snf 1激酶复合物。纯化的激酶复合物是酶活性的判断，其磷酸化的重组蛋白含有Snf 1-响应域的Mig 1蛋白的能力。含有Gal 83或Sip 2作为β亚基的Snf 1激酶复合物显示出相当高的活性水平，而含有Sip 1的酶活性显著降低。纯化的Snf 1酶复合物的蛋白质组成的检查表明，Sip 1蛋白存在于亚化学计量水平。增加SIP 1的基因剂量挽救了在表达Sip 1作为其唯一β亚基的细胞中观察到的乙醇生长缺陷，并增加了从这些细胞中纯化的Snf 1激酶的体外活性。我们的研究表明，Snf 1-Snf 4-Sip 1激酶的活性降低是由于Sip 1积累水平低，而不是Sip 1形式的酶直接磷酸化特定底物的能力有限。
The Snf1 kinase complex of Saccharomyces cerevisiae contains one of three possible beta subunits encoded by either SIP1, SIP2, or GAL83. Snf1 kinase complexes were purified from cells expressing only one of the three beta subunits using a tandem affinity purification tag on the C terminus of the Snf1 protein. The purified kinase complexes were enzymatically active as judged by their ability to phosphorylate a recombinant protein containing the Snf1-responsive domain of the Mig1 protein. The Snf1 kinase complexes containing Gal83 or Sip2 as the beta subunit showed comparable and high levels of activity, whereas the Sip1-containing enzyme was significantly less active. Examination of the protein composition of the purified Snf1 enzyme complexes indicated that the Sip1 protein was present in substoichiometric levels. Increased gene dosage of SIP1 rescued the ethanol growth defect observed in cells expressing Sip1 as their only beta subunit and increased the in vitro activity of Snf1 kinase purified from these cells. Our studies indicate that the reduced activity of Snf1-Snf4-Sip1 kinase is due to low level of Sip1 accumulation rather than a limited ability of the Sip1 form of the enzyme to direct phosphorylation of specific substrates.