Sec12p is the guanine nucleotide exchange factor of Sar1 GTPase and functions at the very upstream in the vesicle budding reactions from the endoplasmic reticulum (ER). We previously identified three yeast loci, RST1, RST2, and RST3, whose mutations suppressed the temperature-sensitive growth of the sec12-4 mutant (Nakano, A. (1996) J. Biochem. (Tokyo) 120, 642-646). In the present study, we cloned the wild-type RST2 gene by complementation of the cold-sensitive phenotype of the rst2-1 mutant. RST2 turned out to be identical to HRR25, a gene encoding a dual-specificity casein kinase I in yeast. The rst2-1 mutation, which is now renamed hrr25-2, was due to the T176I amino acid replacement in the kinase domain. This mutation remedied not only the temperature-sensitive growth but also the defect of ER-to-Golgi protein transport of sec12. Immunoprecipitation of the hemagglutinin-tagged Hrr25-2 protein and a subsequent protein kinase assay showed that the kinase activity of the mutant protein was markedly reduced. The overproduction of another kinase-minus mutant of Hrr25p (Hrr25p K38A) slightly suppressed the growth defect of sec12-4 as well. These observations suggest that the reduction of the kinase activity in the mutant protein is important for the suppression of sec12. We propose that Hrr25p negatively regulates the vesicle budding from the ER.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|