酿酒酵母ssl1突变株分泌羧肽酶Y的胞外加工

Yoichiro Shiba, Kimihisa Ichikawa, Nobufusa Serizawa, Hiroji Yoshikawa
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引用次数: 7

摘要

羧肽酶Y (CPY);电子商务3。4. 16. 1)由PRC1基因编码的酵母液泡蛋白酶。它作为酶原proCPY进入液泡,被液泡酶,蛋白酶a (PrA)和蛋白酶B (PrB)激活。我们之前的研究表明,从酿酒酵母ssl1(溶菌酶超分泌)突变体中有效地分泌活性CPY,该突变体携带含有PRC1基因的多拷贝质粒,该质粒与诱导型GAL10启动子融合。在本研究中,我们检测了携带CPY表达质粒的ssl1突变体的培养上清中PrA和PrB的活性。胞外CPY的n端氨基酸序列与原液泡序列一致。此外,使用蛋白酶抑制剂的研究表明,CPY以前体的形式分泌到培养基中,主要被细胞外PrB激活。即使只有一个PRC1基因拷贝,ssl1突变体也会在培养基中分泌CPY、PrA和PrB。另一方面,无论PRC1基因是否过表达,培养基中均未检测到胞质标记酶葡萄糖-6-磷酸脱氢酶和空泡膜相关酶α-甘露糖苷酶。提示空泡蛋白酶的分泌与ssl1突变或PRC1基因的过表达有关。
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Extracellular processing of carboxypeptidase Y secreted by a Saccharomyces cerevisiae ssl1 mutant strain

Carboxypeptidase Y (CPY; EC 3. 4. 16. 1) encoded by the PRC1 gene is a yeast vacuolar protease. It enters the vacuole as a zymogen, proCPY, which is activated by vacuolar enzymes, proteinase A (PrA) and proteinase B (PrB). We previously showed that active CPY was efficiently secreted from the Saccharomyces cerevisiae ssl1 (supersecretion of lysozyme) mutant carrying a multicopy plasmid which contains the PRC1 gene fused to an inducible GAL10 promoter. In this study, we detected PrA and PrB activities in the culture supernatant of the ssl1 mutant harboring the CPY expression plasmid. The N-terminal amino acid sequence of extracellular CPY coincided with that of the original vacuolar one. Furthermore, studies using protease inhibitors suggested that CPY was secreted into the medium in the form of a precursor and was mainly activated by extracellular PrB. The ssl1 mutant secreted CPY, PrA and PrB into the medium even with a single copy of the PRC1 gene. On the other hand, a cytoplasmic marker enzyme, glucose-6-phosphate dehydrogenase, and a vacuolar membraneassociated enzyme, α-mannosidase, were not detected in the medium, whether the PRC1 gene was overexpressed or not. It is suggested that secretion of vacuolar proteases is caused by characteristics of the ssl1 mutation or overexpression of the PRC1 gene.

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