Isolation and deduced amino acid sequence of the gene encoding gp115, a yeast glycophospholipid-anchored protein containing a serine-rich region.
gp115 is a N- and O-glycosylated protein of Saccharomyces cerevisiae. It is also modified by addition of glycosylphosphatidylinositol, which anchors the protein to the plasma membrane. The gene encoding gp115 (GGP1) has been cloned by a two-step procedure. By an immunoscreening of a yeast genomic DNA library in the expression vector lambda gt11, a 3'-terminal 0.9-kilobase portion of the gene has been isolated and then used as a molecular probe to screen a yeast genomic DNA library in YEp24. In this way, the whole GGP1 gene has been cloned. Its identity with the gp115 gene has been confirmed by gene disruption, which has also indicated that the function of gp115 is not essential for cell viability. The features of the sequence are also entirely consistent with it corresponding to the gp115 gene. The nucleotide sequence of GGP1 predicts a 60-kDa polypeptide, in agreement with the molecular mass of the gp115 precursor detected in sec53 mutant cells at restrictive temperature. Two hydrophobic sequences, one NH2- and the other COOH-terminal were found. The former has the features of the cleavable signal sequence, which allows the entry of proteins in the secretory pathway. The latter could be the signal sequence that has to be removed during the addition of glycosylphosphatidylinositol. The predicted amino acid sequence of gp115 shows 10 sequons for N-glycosylation and a high proportion of serine-threonine residues (22%) that could provide several sites for O-glycosylation. The unusual concentration of 27 serines in the COOH-terminal portion of the protein shares homology with a similar polyserine repeat of the serine repeat antigen (SERA protein) of Plasmodium falciparum. A two-dimensional analysis of the "in vitro" translational product of the GGP1 mRNA has been carried out, allowing the identification of the "in vivo" gp115 precursor in a two-dimensional gel.
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Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
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Lacanà
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Alberghina
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D004271:DNA, Fungal / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D008562:Membrane Glycoproteins; D012331:RNA, Fungal; C030340:elastin microfibril interface located protein