Busnelli Sara, Tripodi Farida, Nicastro Raffaele, Cirulli Claudia, Tedeschi Gabriella, Pagliarin Roberto, Alberghina Lilia, Coccetti Paola
SYSBIO, Centre of Systems Biology, Milan, Italy; Department of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy.
D.I.P.A.V.-Biochemistry, University of Milano, Via Celoria 10, 20133 Milan, Italy.
Biochim Biophys Acta. 2013 Dec;1833(12):3254-3264. doi: 10.1016/j.bbamcr.2013.09.014. Epub 2013 Sep 29.
Snf1, the yeast AMP-activated kinase homolog, regulates the expression of several genes involved in adaptation to glucose limitation and in response to cellular stresses. We previously demonstrated that Snf1 interacts with Swi6, the regulatory subunit of SBF and MBF complexes, and activates CLB5 transcription. Here we report that, in α-factor synchronized cells in 2% glucose, the loss of the Snf1 catalytic subunit impairs the binding of SBF and MBF complexes and the subsequent recruitment of the FACT complex and RNA Polymerase II to promoters of G1-genes. By using an analog-sensitive allele of SNF1, SNF1(as)(I132G), encoding a protein whose catalytic activity is selectively inhibited in vivo by 2-naphthylmethyl pyrazolopyrimidine 1, we show that the inhibition of Snf1 catalytic activity affects the expression of G1-genes causing a delayed entrance into S phase in cells synchronized in G1 phase by α-factor treatment or by elutriation. Moreover, Snf1 is detected in immune complexes of Rpb1, the large subunit of RNA Polymerase II, and is present at both promoters and coding regions of SBF- and MBF-regulated genes 20min after α-factor release, suggesting a direct role for Snf1 in the activation of the G1-regulon transcription.
Snf1是酵母中与AMP激活的蛋白激酶同源的蛋白,它调控着多个参与适应葡萄糖限制及应对细胞应激的基因的表达。我们之前证实Snf1与SBF和MBF复合物的调节亚基Swi6相互作用,并激活CLB5转录。在此我们报告,在2%葡萄糖条件下经α因子同步化处理的细胞中,Snf1催化亚基的缺失会损害SBF和MBF复合物的结合,以及随后FACT复合物和RNA聚合酶II向G1期基因启动子的募集。通过使用SNF1的一个对类似物敏感的等位基因SNF1(as)(I132G),其编码的蛋白在体内可被2-萘甲基吡唑并嘧啶1选择性抑制催化活性,我们发现抑制Snf1催化活性会影响G1期基因的表达,导致经α因子处理或淘洗在G1期同步化的细胞进入S期延迟。此外,在RNA聚合酶II的大亚基Rpb1的免疫复合物中检测到了Snf1,并且在α因子释放20分钟后,Snf1存在于SBF和MBF调控基因的启动子及编码区,这表明Snf1在激活G1调控子转录中具有直接作用。