Mutational analysis of the Saccharomyces cerevisiae SNF1 protein kinase and evidence for functional interaction with the SNF4 protein.

The SNF1 gene of Saccharomyces cerevisiae encodes a protein-serine/threonine kinase that is required for derepression of gene expression in response to glucose limitation. We present evidence that the protein kinase activity is essential for SNF1 function: substitution of Arg for Lys in the putative ATP-binding site results in a mutant phenotype. A polyhistidine tract near the N terminus was found to be dispensable. Deletion of the large region C terminal to the kinase domain only partially impaired SNF1 function, causing expression of invertase to be somewhat reduced but still glucose repressible. The function of the SNF4 gene, another component of the regulatory system, was required for maximal in vitro activity of the SNF1 protein kinase. Increased SNF1 gene dosage partially alleviated the requirement for SNF4. C-terminal deletions of SNF1 also reduced dependence on SNF4. Our findings suggest that SNF4 acts as a positive effector of the kinase but does not serve a regulatory function in signaling glucose availability.     

Roles of the glycogen-binding domain and Snf4 in glucose inhibition of SNF1 protein kinase

The SNF1/AMP-activated protein kinase (AMPK) family is required for adaptation to metabolic stress and energy homeostasis. The gamma subunit of AMPK binds AMP and ATP, and mutations that affect binding cause human disease. We have here addressed the role of the Snf4 (gamma) subunit in regulating SNF1 protein kinase in response to glucose availability in Saccharomyces cerevisiae. Previous studies of mutant cells lacking Snf4 suggested that Snf4 counteracts autoinhibition by the C-terminal sequence of the Snf1 catalytic subunit but is dispensable for glucose regulation, and AMP does not activate SNF1 in vitro. We first introduced substitutions at sites that, in AMPK, contribute to nucleotide binding and regulation. Mutations at several sites relieved glucose inhibition of SNF1, as judged by catalytic activity, phosphorylation of the activation-loop Thr-210, and growth assays, although analogs of the severe human mutations R531G/Q had little effect. We further showed that alterations of Snf4 residues that interact with the glycogen-binding domain (GBD) of the beta subunit strongly relieved glucose inhibition. Finally, substitutions in the GBD of the Gal83 beta subunit that are predicted to disrupt interactions with Snf4 and also complete deletion of the GBD similarly relieved glucose inhibition of SNF1. Analysis of mutant cells lacking glycogen synthase showed that regulation of SNF1 is normal in the absence of glycogen. These findings reveal novel roles for Snf4 and the GBD in regulation of SNF1.     

A family of proteins containing a conserved domain that mediates interaction with the yeast SNF1 protein kinase complex.

The SNF1 protein kinase is required for the regulatory response to glucose starvation in Saccharomyces cerevisiae. SNF1 is a protein serine/threonine kinase that has been widely conserved in both plants and mammals. Previously, we identified SIP1 and SIP2 as proteins that interact with SNF1 in vivo by the two-hybrid system. We have cloned the SIP2 gene and the encoded protein is homologous to SIP1 and to GAL83, which affects glucose repression of the GAL genes. We show that SIP2 and GAL83, like SIP1, co-immunoprecipitate with SNF1 and are phosphorylated in vitro. An 80 amino acid sequence, designated the ASC domain, is highly conserved at the C-termini of all three proteins. We show that this small domain can mediate protein-protein interaction with the SNF1 kinase complex. Thus, SIP1, SIP2 and GAL83 define a family of homologous proteins that are tightly associated with the SNF1 kinase, probably in alternative forms of the complex. Genetic evidence suggests that the three proteins have distinct, but related, functions in the SNF1 pathway, and deletion of GAL83 dramatically reduces SNF1 activity in immune complex assays. We propose that SIP1, SIP2 and GAL83 act as adaptors that promote the activity of SNF1 towards specific targets.     

The SNF1 kinase complex from Saccharomyces cerevisiae phosphorylates the transcriptional repressor protein Mig1p in vitro at four sites within or near regulatory domain 1.

Mig1p is a zinc finger protein required for repression of glucose-regulated genes in budding yeast. On removal of medium glucose, gene repression is relieved via a mechanism that requires the SNF1 protein kinase complex. We show that Mig1p expressed as a glutathione-S-transferase fusion in bacteria is readily phosphorylated by the SNF1 kinase in vitro. Four phosphorylation sites were identified, i.e. Ser-222, Ser-278, Ser-311 and Ser-381. The latter three are exact matches to the recognition motif we previously defined for SNF1 and lie within regions shown to be required for SNF1-dependent derepression and nuclear-to-cytoplasmic translocation.     

Sequence and phylogenetic analysis of the SNF4/AMPK gamma subunit gene from Drosophila melanogaster.

To optimize gene expression under different environmental conditions, many organisms have evolved systems which can quickly up- and down-regulate the activity of other genes. Recently, the SNF1 kinase complex from yeast and the AMP-activated protein kinase complex from mammals have been shown to represent homologous metabolic sensors that are key to regulating energy levels under times of metabolic stress. Using heterologous probing, we have cloned the Drosophila melanogaster homologue of SNF4, the noncatalytic effector subunit from this kinase complex. A sequence corresponding to the partial genomic sequence as well as the full-length cDNA was obtained, and shows that the D. melanogaster SNF4 is encoded in a 1944-bp cDNA representing a protein of 648 amino acids (aa). Southern analysis of Drosophila genomic DNA in concert with a survey of mammalian SNF4 ESTs indicates that in metazoans, SNF4 is a duplicated gene, and possibly even a larger gene family. We propose that one gene copy codes for a short (330 aa) protein, whereas the second locus codes for a longer version (<410 aa) that is extended at the carboxy terminus, as typified by the Drosophila homologue presented here. Phylogenetic analysis of yeast, invertebrate, and multiple mammalian isoforms of SNF4 shows that the gene duplication likely occurred early in the metazoan lineage, as the protein products of the different loci are relatively divergent. When the phylogeny was extended beyond the SNF4 gene family, SNF4 shares sequence similarity with other cystathionine-beta-synthase domain-containing proteins, including IMP dehydrogenase and a variety of uncharacterized Methanococcus proteins.     

酿酒酵母SNF4基因的克隆及生物信息学分析

SNF4基因编码的Snf4p具有调节Snf1复合体的蛋白激酶活性功能,根据已知的SNF4基因序列设计引物扩增获得S.cerevisiae YS2的SNF4基因完整序列。序列分析表明,SNF4基因的开放阅读框为969bp,编码322个氨基酸残基。应用生物信息方法预测其理化性质、疏水性、信号肽、亚细胞定位、活性位点及其高级结构。结果表明:Snf4p为具有一定亲水性的非跨膜胞内稳定酸性蛋白,功能结构域为CBS_pair superfamily结构域,二级结构主要由a-螺旋组成,空间结构是由4个CBS结构域构成两个CBS对围绕形成的二聚体。Snf4p的第一个CBS对区域的β片层结构是Snf1p、Sip2p的β发夹结构结合作用区。     

Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.     

Mutational analysis of the SNF3 glucose transporter of Saccharomyces cerevisiae.

The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. Point mutations affecting the function of the transporter were recovered from the genomes of four snf3 mutants and characterized. Two of the mutations introduced a charged amino acid into the first and second predicted membrane-spanning regions, respectively. The analogs of a bifunctional SNF3-lacZ fusion containing these two mutations were constructed, and the mutant fusion proteins were not localized to the plasma membrane, as judged by immunofluorescence microscopy. The third mutation produced a valine-to-isoleucine substitution in hydrophobic region 8, and the corresponding mutant fusion protein was correctly localized. The finding that this conservative change causes a transport defect is consistent with the possibility that this transmembrane region, which could exist as an amphipathic alpha-helix, forms part of the glucose channel through the membrane. The fourth snf3 allele harbored an ochre mutation midway through the coding sequence. We have also constructed mutations in the cloned SNF3 gene. A major difference between the yeast SNF3 protein and mammalian glucose transporters is the presence in the SNF3 protein of an additional 303 amino acids at the C terminus. Analysis of a series of C-terminal deletions and fusions to lacZ showed that this C-terminal region is important, but not essential, for transport function. We also report the genetic mapping of the SNF3 locus on the left arm of chromosome IV.     

ADP regulates SNF1, the Saccharomyces cerevisiae homolog of AMP-activated protein kinase

The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.     

Physiological characterization of glucose repression in the strains with SNF1 and SNF4 genes deleted

We investigated the effect of Snf1 kinase and its regulatory subunit Snf4 on the regulation of glucose and galactose metabolism in the yeast Saccharomyces cerevisiae by physiologically characterizing Deltasnf1, Deltasnf4 and Deltasnf1Deltasnf4 in CEN.PK background in glucose and glucose-galactose-mixture batch cultivations. The main result of this study showed that delayed induction of galactose catabolism was SNF1 or SNF4 gene deletion specific. In comparison to the reference strain, growth delay on galactose was found to last 2.4 times (7 h), 3.1 times (10.5 h) and 9.6 times (43 h) longer for the Deltasnf4, Deltasnf1 and Deltasnf1Deltasnf4 strains, respectively. The maximum specific growth rates on galactose were determined to be two to three times lower for the recombinant strains compared to the reference strain (0.13 h(-1)) and were found to be 0.07, 0.08 and 0.04 h(-1) for the Deltasnf1, Deltasnf4 and Deltasnf1Deltasnf4 strains, respectively. The study showed that Snf1 kinase was not solely responsible for the derepression of galactose metabolism.     

Interaction of SNF1 protein kinase with its activating kinase Sak1

The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change.     

REG1 binds to protein phosphatase type 1 and regulates glucose repression in Saccharomyces cerevisiae.

Protein phosphatase type 1 (PP1) is encoded by GLC7, an essential gene in Saccharomyces cerevisiae. The GLC7 phosphatase is required for glucose repression and appears to function antagonistically to the SNF1 protein kinase. Previously, we characterized a mutation, glc7-T152K, that relieves glucose repression but does not interfere with the function of GLC7 in glycogen metabolism. We proposed that the mutant GLC7T152K phosphatase is defective in its interaction with a regulatory subunit that directs participation of PP1 in the glucose repression mechanism. Here, we present evidence that REG1, a protein required for glucose repression, is one such regulatory subunit. We show that REG1 is physically associated with GLC7. REG1 interacts with GLC7 strongly and specifically in the two-hybrid system, and REG1 and GLC7 fusion proteins co-immunoprecipitate from cell extracts. Moreover, overexpression of a REG1 fusion protein suppresses the glc7-T152K mutant defect in glucose repression. This and other genetic evidence indicate that the two proteins function together in regulating glucose repression. These results suggest that REG1 is a regulatory subunit of PP1 that targets its activity to proteins in the glucose repression regulatory pathway.