Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe.
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.
Tabuchi
M
M
Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Miki-cho, Japan.
Iwaihara
O
O
Ohtani
Y
Y
Ohuchi
N
N
Sakurai
J
J
Morita
T
T
Iwahara
S
S
Takegawa
K
K
D001483:Base Sequence; D001692:Biological Transport; D002268:Carboxypeptidases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D043402:Cathepsin A; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D011498:Protein Precursors; D002268:Carboxypeptidases; D043402:Cathepsin A