Regulation of inositol transport in Saccharomyces cerevisiae involves inositol-induced changes in permease stability and endocytic degradation in the vacuole.
Uptake of inositol by Saccharomyces cerevisiae is mediated by a specific inositol permease encoded by the ITR1 gene. Removal of inositol from the growth medium results in an increase in ITR1 mRNA abundance. The increase in ITR1 mRNA is accompanied by an increase in de novo synthesis of the Itr1 permease leading to an increased capacity for uptake. When inositol is added to the growth medium inactivation of uptake activity occurs, and both transcription of ITR1 and uptake activity are repressed to a basal level of function. The transcriptional regulation of ITR1 depends on the INO2, INO4, and OPI1 genes. In addition, repression is also achieved by regulation of ITR1 expression at the post-translational level. In this study, we show that there is a change in the stability of the Itr1 permease after the addition of inositol to the growth medium. Immunoblot analysis using a monoclonal antibody against an epitope attached to the Itr1 permease showed that the addition of inositol causes a dramatic increase in the rate of degradation of the permease. After the repressed (basal) level is achieved, turnover continues to be rapid. The increased rate of degradation was also observed in strains with mutations that block conjugation to ubiquitin. Degradation was not observed in strains defective in the END3/END4 endocytic pathway or in the production of vacuolar proteases (PEP4). Thus, inactivation of the Itr1 permease is accompanied by endocytic internalization followed by degradation in the vacuole. Inactivation may be a separate process that precedes and signals endocytic degradation. Since the end3/end4 mutations did not affect uptake activity under derepressed conditions, endocytosis is not required for normal inositol uptake.
Lai
K
K
Department of Biological Sciences, University of Maryland, Baltimore 21228.
Bolognese
C P
CP
Swift
S
S
McGraw
P
P
D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004705:Endocytosis / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007294:Inositol / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014158:Transcription, Genetic; D014617:Vacuoles / Q000201:enzymology*
D002352:Carrier Proteins; D017931:DNA Primers; D005656:Fungal Proteins; C069143:ITR1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D007294:Inositol