N-Terminal Mutations Modulate Yeast SNF1 Protein Kinase Function

• Published in: Genetics (Austin) 1992-11, Vol.132 (3), p.639-650
• Main Authors: Estruch, F, Treitel, M. A, Yang, X, Carlson, M
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; AMP-Activated Protein Kinases; Base Sequence; Biological and medical sciences; Carrier Proteins; Chromatography, Gel; Classical genetics, quantitative genetics, hybrids; Enzyme Activation; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Genes; Genes, Dominant; Genetics of eukaryotes. Biological and molecular evolution; Immunologic Techniques; Investigations; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Phosphorylation; Protein Kinases - metabolism; Protein Processing, Post-Translational; Protein-Serine-Threonine Kinases - genetics; Protein-Serine-Threonine Kinases - metabolism; Proteins; Recombinant Fusion Proteins - biosynthesis; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; Thallophyta, bryophyta; Transcription Factors - metabolism; Vegetals; Yeast
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/132.3.639

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.