Heterologous expression and characterization of Schizosaccharomyces pombe vacuolar carboxypeptidase Y in Saccharomyces cerevisiae.
To investigate the intracellular transport mechanism of the vacuolar carboxypeptidase of Schizosaccharomyces pombe (SpCPY), SpCPY was expressed in Saccharomyces cerevisiae and its biosynthesis and sorting were examined. When Sac. cerevisiae prc1Delta, devoid of intrinsic (Sc) CPY activity, was transformed with a plasmid carrying the Sch. pombe cpy1(+) gene, CPY activity was restored. Pulse-chase experiments revealed that SpCPY is initially synthesized in a pro-precursor form and then converted to a heterodimer, the mature form, in Sac. cerevisiae cells. SpCPY was not processed into intermediate or mature forms in pep4 mutant cells, indicating that SpCPY was proteolytically cleaved in a PEP4-dependent manner in Sac. cerevisiae. Several vps mutants, which are defective in vacuolar protein-sorting, exhibited a defect in the maturation of SpCPY. Moreover, the maturation of SpCPY was severely inhibited in a vps10 strain, although the pro- segment of SpCPY does not contain a QRPL-like sequence, which is the putative targeting signal of ScCPY. When SpCPY was expressed in a wild-type strain, more than 90% of ScCPY was normally sorted to the vacuole, indicating that SpCPY does not compete with ScCPY for vacuolar sorting. In contrast, expression of SpCPY resulted in a missorting of a ScCPY-invertase fusion protein to the cell surface. These results suggested that there are two different binding sites for SpCPY and ScCPY on Vps10p and that the binding of SpCPY to Vps10p interferes with the binding of a ScCPY-invertase fusion protein.
Takegawa
Kaoru
K
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. takegawa@ag.kagawa-u.ac.jp
Tokudomi
Sanae
S
Bhuiyan
M Shah Alam
MS
Tabuchi
Mitsuaki
M
Fujita
Yasuko
Y
Iwaki
Tomoko
T
Utsumi
Shigeru
S
Tanaka
Naotaka
N
D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism*; D043402:Cathepsin A; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics*; D017384:Sequence Deletion; D014617:Vacuoles / Q000201:enzymology
D011994:Recombinant Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A