In vivo phosphorylation of the purine/cytosine permease from the plasma membrane of the yeast Saccharomyces cerevisiae.
The purine/cytosine permease, encoded by the FCY2 gene, is a carrier located in the plasma membrane of the yeast Saccharomyces cerevisiae. Polyclonal antibodies were raised against two peptides that corresponded to the sub-N-terminal and C-terminal sequences of the putative protein deduced from the FCY2 gene. Immunoprecipitation experiments performed with protein extracts labelled in vivo with 35S showed that purine/cytosine permease is specifically detected as a broad and diffuse band. The apparent molecular mass of this protein was 45-50 kDa. By means of in vivo pulse/chase 35S-labelling experiments, we observed a slight increase in the apparent molecular mass of purine/cytosine permease during the chase. This shift in electrophoretic mobility of the protein suggested a post-translational modification. This molecular mass increase was eliminated by alkaline phosphatase treatment of the immunoprecipitate, which strongly suggested phosphorylation of the carrier. This proposal was confirmed by in vivo [32P]P(i) labelling and immunoprecipitation of purine/cytosine permease with purified anti-(sub-N-terminal peptide) IgG or anti-(C-terminal peptide) IgG. Phosphoamino acid analysis indicated that phosphorylation occurred on seryl residues of purine/cytosine permease. By means of thermosensitive secretory-pathway-mutant strains, we demonstrated that purine/cytosine permease phosphorylation occurred either between the Golgi apparatus and the plasma membrane or in the plasma membrane itself.
Pinson
B
B
Institut de Biochimie et Génétique Cellulaires du Centre National de la Recherche Scientifique, Bordeaux, France.
Pillois
X
X
Brèthes
D
D
Chevallier
J
J
Napias
C
C
D000595:Amino Acid Sequence; D000906:Antibodies; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D005800:Genes, Fungal; D015151:Immunoblotting; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D033702:Nucleobase Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D000906:Antibodies; D002352:Carrier Proteins; C043181:FCY2 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033702:Nucleobase Transport Proteins; D010446:Peptide Fragments; D010748:Phosphopeptides; D029701:Saccharomyces cerevisiae Proteins