Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase.

The SNF4 gene is required for expression of glucose-repressible genes in response to glucose deprivation in Saccharomyces cerevisiae. Previous evidence suggested that SNF4 is functionally related to SNF1, another essential gene in this global regulatory system that encodes a protein kinase. Increased SNF1 gene dosage partially compensates for a mutation in SNF4, and the SNF4 function is required for maximal SNF1 protein kinase activity in vitro. We have cloned SNF4 and identified its 1.2-kilobase RNA, which is not regulated by glucose repression. A 36-kilodalton SNF4 protein is predicted from the nucleotide sequence. Disruption of the chromosomal SNF4 locus revealed that the requirement for SNF4 function is less stringent at low temperature (23 degrees C). A bifunctional SNF4-lacZ gene fusion that includes almost the entire SNF4 coding sequence was constructed. The fusion protein was shown by immunofluorescence microscopy to be distributed throughout the cell, with partial localization to the nucleus. The SNF4-beta-galactosidase protein coimmunoprecipitated with the SNF1 protein kinase, thus providing evidence for the physical association of the two proteins.     

N-Terminal Mutations Modulate Yeast Snf1 Protein Kinase Function

The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-delta N kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-delta N protein showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell.     

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.     

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.     

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.     

Identification of a sucrose nonfermenting-1-related protein kinase in sugar beet (Beta vulgaris L.)

A 154 bp polymerase chain reaction product, SBKIN154, showing 76–83% sequence identity with sucrose nonfermenting-1 (SNF1)-related protein kinase nucleotide sequences from other plant species was amplified from sugar beet storage root RNA. Southern blot analysis using SBKIN154 as a hybridisation probe suggested that sugar beet contains either a single-copy SNF1-related gene or a small gene family of highly conserved genes. An antibody raised to a heterologously-expressed fusion of the rye SNF1-related protein kinase, RKIN1, and maltose binding protein, recognised a protein of the expected size (M_r approx. 60,000) on western blots of storage root, stalk, leaf and root extracts. Measurements of SNF1-related activity were made using a specific peptide (SAMS) phosphorylation assay. Activity was highest (0.38 nmol min^-1 mg^-1 protein) in developing storage roots and lowest (0.035 nmol min^-1 mg^-1) in fibrous roots.     

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.     

CHB2, a member of the SWI3 gene family,is a global regulator in Arabidopsis

The SWI/SNF complex is an ATP-dependent chromatin remodeling complex that plays an important role in the regulation of eukaryotic gene expression. Very little is known about the function of SWI/SNF complex in plants compared with animals and yeast. SWI3 is one of the core components of the SWI/SNF chromatin remodeling complexes in yeast. We have identified a putative SWI3-like cDNA clone, CHB2 (AtSWI3B), from Arabidopsis thaliana by screening the expressed sequence tag database. CHB2 encodes a putative protein of 469 amino acids and shares 23% amino acid sequence identity and 64% similarity with the yeast SWI3. The Arabidopsis genome contains four SWI3-like genes, namely CHB1 (AtSWI3A), CHB2 (AtSWI3B), CHB3 (AtSWI3C) and CHB4 (AtSWI3D). The expression of CHB2, CHB3 and CHB4 mRNA was detected in all tissues analyzed by RT-PCR. The expression of CHB1 mRNA, however, could not be detected in the siliques, suggesting that there is differential expression among CHB genes in different Arabidopsis tissues. To investigate the role of CHB2 in plants, Arabidopsis plants were transformed with a gene construct comprising a CHB2 cDNA in the antisense orientation driven by the CaMV 35S promoter. Repression of CHB2 expression resulted in pleiotropic developmental abnormalities including abnormal seedling and leaf phenotypes, dwarfism, delayed flowering and no apical dominance, suggesting a global role for CHB2 in the regulation of gene expression. Our results indicate that CHB2 plays an essential role in plant growth and development.     

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.     

Characterization of the DNA-binding activity of GCR1: in vivo evidence for two GCR1-binding sites in the upstream activating sequence of TPI of Saccharomyces cerevisiae.

GCR1 gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae. Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTPI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcr1 deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcr1 mutant strain. These data demonstrate that the UAS of TPI contains two GCR1-binding sites which are occupied in vivo. Furthermore, adjacent RAP1/GRF1/TUF- and REB1/GRF2/QBP/Y-binding sites in UASTPI were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO1, PYK, and ADH1, all of which are dependent on GCR1 gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence elements found in front of the translational component genes TEF1, TEF2, and CRY1. Taken together, these experiments have allowed us to propose a consensus GCR1-binding site which is 5'-(T/A)N(T/C)N(G/A)NC(T/A)TCC(T/A)N(T/A)(T/A)(T/G)-3'.