ASPEX Division, Research Center, Asahi Glass Co., Ltd., 1150 Hazawa-cho, Kanagawa, 221-8755, Japan.
Appl Microbiol Biotechnol. 2011 Apr;90(1):203-13. doi: 10.1007/s00253-010-3031-3. Epub 2010 Dec 14.
Schizosaccharomyces pombe carboxypeptidase Y (CPY) is synthesized as a zymogen and transported into the vacuole where maturation and activation occurs. The 110-kDa S. pombe CPY precursor is processed twice and finally converted to a mature form consisting of polypeptides of approximately 19 and 32 kDa linked by a single disulfide bond. In Saccharomyces cerevisiae, maturation of CPY occurs mostly through the activity of vacuolar aspartyl protease Pep4p, whereas a Pep4p homolog has not been found in the S. pombe genome database. Based on analysis of protease-deficient mutants, we found that S. pombe CPY was not able to be processed or activated in isp6Δpsp3Δ double disruptants. Both Isp6p and Psp3p are subtilase-type serine proteases with related sequences. Moreover, alkaline phosphatase of S. pombe was found to be localized at the vacuolar membrane and was also unprocessed in isp6Δpsp3Δ double disruptants. Vacuolar localization of GFP-fused Isp6p and Psp3p was determined by fluorescence microscopy. These results suggest that the two serine proteases Isp6p and Psp3p are functional in the vacuole and are involved in proteolytic processing of vacuolar proteins.
裂殖酵母羧肽酶 Y（CPY）作为酶原合成并被运输到液泡中，在那里进行成熟和激活。110 kDa 的裂殖酵母 CPY 前体经过两次加工，最终转化为一种成熟形式，由大约 19 和 32 kDa 的多肽通过单个二硫键连接而成。在酿酒酵母中，CPY 的成熟主要通过液泡天冬氨酸蛋白酶 Pep4p 的活性发生，而在裂殖酵母基因组数据库中未发现 Pep4p 同源物。基于对蛋白酶缺陷突变体的分析，我们发现，在 isp6Δpsp3Δ 双缺失突变体中，CPY 无法进行加工或激活。Isp6p 和 Psp3p 都是具有相似序列的枯草杆菌蛋白酶型丝氨酸蛋白酶。此外，裂殖酵母碱性磷酸酶被发现定位于液泡膜上，并且在 isp6Δpsp3Δ 双缺失突变体中也未进行加工。通过荧光显微镜确定 GFP 融合的 Isp6p 和 Psp3p 的液泡定位。这些结果表明，两种丝氨酸蛋白酶 Isp6p 和 Psp3p 在液泡中具有功能，并参与液泡蛋白的蛋白水解加工。
