Key role of Ser562/661 in Snf1-dependent regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis - PubMed (original) (raw)

Comparative Study

Key role of Ser562/661 in Snf1-dependent regulation of Cat8p in Saccharomyces cerevisiae and Kluyveromyces lactis

Godefroid Charbon et al. Mol Cell Biol. 2004 May.

Abstract

Utilization of nonfermentable carbon sources by Kluyveromyces lactis and Saccharomyces cerevisiae requires the Snf1p kinase and the Cat8p transcriptional activator, which binds to carbon source-responsive elements of target genes. We demonstrate that KlSnf1p and KlCat8p from K. lactis interact in a two-hybrid system and that the interaction is stronger with a kinase-dead mutant form of KlSnf1p. Of two putative phosphorylation sites in the KlCat8p sequence, serine 661 was identified as a key residue governing KlCat8p regulation. Serine 661 is located in the middle homology region, a regulatory domain conserved among zinc cluster transcription factors, and is part of an Snf1p consensus phosphorylation site. Single mutations at this site are sufficient to completely change the carbon source regulation of the KlCat8p transactivation activity observed. A serine-to-glutamate mutant form mimicking constitutive phosphorylation results in a nearly constitutively active form of KlCat8p, while a serine-to-alanine mutation has the reverse effect. Furthermore, it is shown that KlCat8p phosphorylation depends on KlSNF1. The Snf1-Cat8 connection is evolutionarily conserved: mutation of corresponding serine 562 of ScCat8p gave similar results in S. cerevisiae. The enhanced capacity of ScCat8S562E to suppress the phenotype caused by snf1 strengthens the hypothesis of direct phosphorylation of Cat8p by Snf1p. Unlike that of S. cerevisiae ScCAT8, KlCAT8 transcription is not carbon source regulated, illustrating the prominent role of posttranscriptional regulation of Cat8p in K. lactis.

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Figures

FIG. 1.

FIG. 1.

Restriction and domain map of KlCat8p and its deletion alleles. The two black boxes represent the zinc cluster and the downstream coiled-coil domains, highly conserved in more than 100 zinc cluster proteins. The grey line represents the middle homology region found in more than 50 zinc cluster proteins. The small black squares cover regions that are highly conserved only among the three known yeast Cat8p homologues. The stars indicate the positions of the eight motifs conserved in zinc cluster proteins as described by Poch (25). Serines 661 and 871 are positioned on the KlCat8p sequence. Grey arrows indicate the sites used in the internal deletion Δ48-3405, and black arrows point to the C-terminal deletion Δ3204-4335.

FIG. 2.

FIG. 2.

Heterospecific complementation of an Sccat8 mutant with deletion and serine mutant alleles of KlCat8p. (A) Strains CEN.NB1-1A (Sccat8) and CEN.PK2-1C (ScCAT8) were transformed with pEG202-based plasmids expressing LexAp, LexA-KlCat8, LexA-KlCat8Δ3204-4335p, and LexA-KlCat8Δ48-3405p and streaked on selective culture medium supplemented with glycerol. (B) Strain CEN.NB1-1A was transformed with pGBT9-based plasmids expressing Gal4p, Gal4-KlCat8p, Gal4-KlCat8Δ48-3405p, Gal4-KlCat8S661Ap, Gal4-KlCat8S871Ap, Gal4-KlCat8S661Ep, Gal4-ScCat8p, Gal4-ScCat8S562Ap, and Gal4-ScCat8S562Ep. Transformants were dropped in three successive 10-fold dilutions on selective culture medium supplemented with glycerol.

FIG. 3.

FIG. 3.

Northern blot analysis of KlICL1 and KlCAT8 expression in K. lactis strains harboring various alleles of KlCAT8. Total RNA was extracted from strains JA6 (wt), yGC661A (A), yGC661E (E), yIG8 (Δ), and yIG2 (klmig1) grown in minimal medium containing 2% glucose, 3% galactose-raffinose, or 3% glycerol or ethanol as the carbon source. Each RNA sample (10 μg) was electrophoresed on an agarose gel in the presence of formaldehyde, blotted onto a nylon membrane, and hybridized to KlICL1, KlCAT8, and KlACT1 probes.

FIG. 4.

FIG. 4.

Potential Snf1p recognition sites in homologues of Cat8p. The consensus Snf1p recognition sequence is shown: Φ, hydrophobic residue (L > F = I = M > V in position −5 and L > I > F > M > V in position +4). Potential Snf1p recognition sites in Cat8p homologues are aligned and numbered when possible according to the position of the phosphorylatable serine. The alanine and glutamic acid substitutions resulting from site-directed mutagenesis are underlined.

FIG. 5.

FIG. 5.

Suppression of the ethanol growth defect of an scsnf1_Δ strain by overexpression of different alleles of ScCAT8. Y14311 (Δ_snf1) was transformed with plasmids YEp351 (empty plasmid) and plasmids YEpCAT8, YEpCAT8S562A, and YEpCAT8S562E, respectively, expressing ScCat8p, ScCat8S562Ap, and ScCat8S562Ep. pGBT9SNF1, expressing KlSnf1p, was transformed into Y14311 as a positive control. Transformants were dropped at three different dilutions onto minimal medium containing 3% ethanol as a carbon source and glucose-rich medium as a positive growth control.

FIG. 6.

FIG. 6.

Effect of a serine 661 mutation in KlCAT8 on growth in ethanol. Strain JA6 (wild type), yIG8 (Δ_klcat8_), yGC661A (_klcat8_-S661A), and yGC661E (_KlCAT8_-S661E) cells were pregrown in minimal medium containing 2% glucose and diluted in minimal medium containing 3% ethanol to an OD600 of 0.1, and the OD600 was monitored for 130 h.

FIG. 7.

FIG. 7.

Western blot analysis of KlCat8p posttranslational modification. Cells were pregrown in 2% glucose and shifted for 2 h to 2% glucose (G) or 3% ethanol (E). (A) Wild-type (WT) strain JA6 was transformed with a multicopy plasmid (HApGID1) expressing HA-KlCat8p. (B) Wild-type strain JA6 and Δ_klsnf1_ mutant strain JSD1R4 were transformed with a multicopy plasmid (HApGID1) expressing HA-KlCat8p.

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References

    1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1994. Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y.
    1. Breunig, K. D., M. Bolotin-Fukuhara, M. M. Bianchi, D. Bourgarel, C. Falcone, I. Ferrero, L. Frontali, P. Goffrini, J. J. Krijger, C. Mazzoni, C. Milkowski, H. Y. Steensma, M. Wesolowski-Louvel, and A. M. Zeeman. 2000. Regulation of primary carbon metabolism in Kluyveromyces lactis. Enzyme Microb. Technol. 26:771-780. - PubMed
    1. Breunig, K. D., and P. Kuger. 1987. Functional homology between the yeast regulatory proteins GAL4 and LAC9: LAC9-mediated transcriptional activation in Kluyveromyces lactis involves protein binding to a regulatory sequence homologous to the GAL4 protein-binding site. Mol. Cell. Biol. 7:4400-4406. - PMC - PubMed
    1. Carlson, M., B. C. Osmond, and D. Botstein. 1981. Mutants of yeast defective in sucrose utilization. Genetics 98:25-40. - PMC - PubMed
    1. Caspary, F., A. Hartig, and H.-J. Schüller. 1997. Constitutive and carbon source-responsive promoter elements are involved in the regulated expression of the Saccharomyces cerevisiae malate synthase gene MLS1. Mol. Gen. Genet. 255:619-627. - PubMed

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