Goller S P, Schoisswohl D, Baron M, Parriche M, Kubicek C P
Institute for Biochemical Technology and Microbiology, Technische Universität Wien, A-1060 Vienna, Austria.
Appl Environ Microbiol. 1998 Sep;64(9):3202-8. doi: 10.1128/AEM.64.9.3202-3208.1998.
Cell extracts of Trichoderma reesei exhibited dibasic endopeptidase activity toward the carboxylic side of KR, RR, and PR sequences. This activity was stimulated by the presence of Ca2+ ions and localized in vesicles of low bouyant density; it therefore exhibited some similarity to yeast Kex2. Analytical chromatofocusing revealed a single peak of activity. The dibasic endopeptidase activity was strongly and irreversibly inhibited in vitro as well as in vivo by 1 mM p-amidinophenylmethylsulfonyl fluoride (pAPMSF) but not by PMSF at concentrations up to 5 mM. We therefore used pAPMSF to study the role of the dibasic endopeptidase in the secretion of protein by T. reesei. Secretion of xylanase I (proprotein processing sequence -R-R- downward arrow-R- downward arrow-A-) and xylanase II (-K-R- downward arrow-Q-) was strongly inhibited by 1 mM pAPMSF, and a larger, unprocessed enzyme form was detected intracellularly under these conditions. Secretion of cellobiohydrolase II (CBH II; -E-R- downward arrow-Q-) was only slightly inhibited by pAPMSF, and no accumulation of unprocessed precursors was detected. In contrast, secretion of CBH I (-R-A- downward arrow-Q-) was stimulated by pAPMSF addition, and a simultaneous decrease in the concentration of intracellular CBH I was detected. Similar experiments were also carried out with a single heterologous protein, ShBLE, the phleomycin-binding protein from Streptoalloteichus hindustanus, fused to a series of model proprotein-processing sequences downstream of the expression signals of the Aspergillus nidulans gpdA promoter. Consistent with the results obtained with homologous proteins, pAPMSF inhibited the secretion of ShBLE with fusions containing dibasic (RK and KR) target sequences, but it even stimulated secretion in fusions to LR, NHA, and EHA target sequences. Addition of 5 mM PMSF, a nonspecific inhibitor of serine protease, nonspecifically inhibited the secretion of heterologous proteins from fusions bearing the NHA and LR targets. These data point to the existence of different endoproteolytic proprotein processing enzymes in T. reesei and demonstrate that dibasic processing is obligatory for the secretion of the proproteins containing this target.
里氏木霉的细胞提取物对KR、RR和PR序列的羧基侧表现出二肽内切酶活性。这种活性受Ca2+离子的存在刺激，并定位于低浮力密度的囊泡中；因此它与酵母Kex2有一些相似之处。分析性色谱聚焦显示有一个单一的活性峰。二肽内切酶活性在体外和体内均被1 mM对脒基苯甲基磺酰氟（pAPMSF）强烈且不可逆地抑制，但在浓度高达5 mM的苯甲基磺酰氟（PMSF）作用下则不会。因此，我们使用pAPMSF来研究二肽内切酶在里氏木霉蛋白质分泌中的作用。1 mM pAPMSF强烈抑制木聚糖酶I（前体蛋白加工序列 -R-R- 向下箭头 -R- 向下箭头 -A-）和木聚糖酶II（-K-R- 向下箭头 -Q-）的分泌，并且在这些条件下细胞内检测到更大的、未加工的酶形式。纤维二糖水解酶II（CBH II；-E-R- 向下箭头 -Q-）的分泌仅受到pAPMSF的轻微抑制，并且未检测到未加工前体的积累。相反，添加pAPMSF刺激了CBH I（-R-A- 向下箭头 -Q-）的分泌，同时检测到细胞内CBH I浓度的下降。还对一种单一的异源蛋白ShBLE进行了类似实验，ShBLE是来自印度斯坦链霉菌的博来霉素结合蛋白，与一系列模型前体蛋白加工序列融合在构巢曲霉gpdA启动子表达信号下游。与同源蛋白的结果一致，pAPMSF抑制了含有二肽（RK和KR）靶序列融合体的ShBLE分泌，但它甚至刺激了与LR、NHA和EHA靶序列融合体的分泌。添加5 mM PMSF（一种丝氨酸蛋白酶的非特异性抑制剂）非特异性地抑制了带有NHA和LR靶标的融合体中异源蛋白的分泌。这些数据表明里氏木霉中存在不同的内切蛋白前体加工酶，并证明二肽加工对于含有该靶标的前体蛋白的分泌是必不可少的。
