Abstract
The expression of the ATPase gene (PMA1) is regulated by glucose (Rao, R., Drummond-Barbosa, D., and Slayman, C. W. (1993) Yeast 9, 1075-1084) and by the TUF/RAP1/GRF1 transcription factor (Capieaux, E., Vignais, M.-L., Sentenac, A., and Goffeau, A. (1989) J. Biol. Chem. 264, 7437-7446). In this work, we describe the isolation of mutations on seven genes that affect the levels of ATPase. One of these genes (APA1) was cloned by complementation and shown to encode a protein with six putative transmembrane stretches. Expression of APA1 gene is regulated by the carbon source and requires the protein GCR1. Deletion of APA1 causes a defective regulation of the PMA1 expression by glucose but has not noticeable effect on the expression of other TUF-regulated genes. Nevertheless the expression of glucose-repressible HXT3 and SNF3 genes is significantly reduced. These results suggest a model in which APA1 acts on a glucose-signaling pathway that controls the expression of several genes that are transcriptionally regulated by glucose.
Highlights
From the Departamento de Bioquimica, Facultad de Medicina, Uniuersidad Autonoma de MadridZ, nstituto de Znvestigaciones Biomedicas del Consejo Superior de Znuestigacioned Cientificas, ArturoDuperier, 4, 28029 Madrid, Spain
The expression otfheATPase gene ( P M A l )is regulated We have initiated a search for mutants affectedin t h e reguby glucose
264,7437-7446).In this work, we describe thiseolationof A P A l gene and data indicating that APAl could function on the mutations onseven genes that affect thleevels ofATPase. regulation of the glucose-dependent expressionof P M A l
Summary
Whenindicated the glucose medium was buffered with 50 mM Mes' adjusted to pH 6.0 with Tris (SD6.0)or with 50 mM succinic acid adjusted topH3.0 with Tris (SD3.0).Hygromicin B resistance was tested in YPD (1%yeast extract, 2% peptone, and 2% glucose)supple-. For each culture a drop of-10' cells was plated on hygromicin B media. Mutants growing slowly on acid media were selectedfor further analysis. Cloning and Sequence Analysis of APAl Gene-Yeast carrying the transcriptional level, glucose increases the synthesis of P M A l apal-3 mutant allele was transformed (Ito et al, 1983)with 50 pg of mRNA (Raoet al., 1993).This regulation seems to be controlledDNA from a YCp50-basedgenomic library (Rose et al, 1987). After 4 days at 30 "C only six colonies grewon acidic media. 'The abbreviations used are: Mes, 4-morpholineethanesulfonic acid; Fax: 34-1-5854587 S n l l ; A,' XhoI; X h l . pRS316 is n single-copy plasmid (Sikomki :and Hirter. 19x91.YICp.75'2 is a multicopy vector 11lill rt nl.. 1986) and YEpR57R is a multicopy vector usrtl fnr ImrX fusion I Myers r t nl.. 1986). p \ l G l l 9 \vas usrd to construct yrnst crlls drlrted for Al.l.11
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