Vacuolar protein sorting in fission yeast: Cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe

Mitsuaki Tabuchi, Osamu Iwaihara, Yoshihiko Ohtani, Nobuhiro Ohuchi, Jun Ichiro Sakurai, Toshie Morita, Shojiro Iwahara, Kaoru Takegawa

Research output: Contribution to journalArticle

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Abstract

PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single- polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.

Original languageEnglish
Pages (from-to)4179-4189
Number of pages11
JournalJournal of bacteriology
Volume179
Issue number13
DOIs
Publication statusPublished - Jul 1997
Externally publishedYes

Fingerprint

Cathepsin A
Schizosaccharomyces
Protein Transport
Organism Cloning
Amino Acids
Saccharomyces cerevisiae
Proteins
beta-Fructofuranosidase
Peptides
Gene Fusion
Vacuoles
Oligosaccharides
Glycosylation
Sodium Dodecyl Sulfate
Organelles
Open Reading Frames
Genes
Polyacrylamide Gel Electrophoresis
Polymerase Chain Reaction
Enzymes

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

Vacuolar protein sorting in fission yeast : Cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. / Tabuchi, Mitsuaki; Iwaihara, Osamu; Ohtani, Yoshihiko; Ohuchi, Nobuhiro; Sakurai, Jun Ichiro; Morita, Toshie; Iwahara, Shojiro; Takegawa, Kaoru.

In: Journal of bacteriology, Vol. 179, No. 13, 07.1997, p. 4179-4189.

Research output: Contribution to journalArticle

Tabuchi, Mitsuaki ; Iwaihara, Osamu ; Ohtani, Yoshihiko ; Ohuchi, Nobuhiro ; Sakurai, Jun Ichiro ; Morita, Toshie ; Iwahara, Shojiro ; Takegawa, Kaoru. / Vacuolar protein sorting in fission yeast : Cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. In: Journal of bacteriology. 1997 ; Vol. 179, No. 13. pp. 4179-4189.
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abstract = "PCR was used to isolate a carboxypeptidase Y (CPY) homolog gene from the fission yeast Schizosaccharomyces pombe. The cloned S. pombe cpy1+ gene has a single open reading frame, which encodes 950 amino acids with one potential N-glycosylation site. It appears to be synthesized as an inactive pre-pro protein that likely undergoes processing following translocation into appropriate intracellular organelles. The C-terminal mature region is highly conserved in other serine carboxypeptidases. In contrast, the N-terminal pro region containing the vacuolar sorting signal in CPY from Saccharomyces cerevisiae shows fewer identical residues. The pro region contains two unusual repeating sequences; repeating sequence I consists of seven contiguous repeating segments of 13 amino acids each, and repeating sequence II consists of seven contiguous repeating segments of 9 amino acids each. Pulse-chase radiolabeling analysis revealed that Cpy1p was initially synthesized in a 110-kDa pro-precursor form and via the 51-kDa single- polypeptide-chain intermediate form which has had its pro segment removed is finally converted to a heterodimer, the mature form, which is detected as a 32-kDa protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Like S. cerevisiae CPY, S. pombe Cpy1p does not require the N-linked oligosaccharide moiety for vacuolar delivery. To investigate the vacuolar sorting signal of S. pombe Cpy1p, we have constructed cpy1+-SUC2 gene fusions that direct the synthesis of hybrid proteins consisting of N-terminal segments of various lengths of S. pombe Cpy1p fused to the secreted enzyme S. cerevisiae invertase. The N-terminal 478 amino acids of Cpy1 are sufficient to direct delivery of a Cpy1-Inv hybrid protein to the vacuole. These results showed that the pro peptide of Cpy1 contains the putative vacuolar sorting signal.",
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