Wolf D H, Ehmann C
Eur J Biochem. 1979 Aug 1;98(2):375-84. doi: 10.1111/j.1432-1033.1979.tb13197.x.
Yeast mutant lacking proteinase B activity have been isolated [Wolf, D. H. and Ehmann, C. (1978) FEBS Lett. 92, 121--124]. One of these mutants (HP232) is characterized in detail. Absence of the vacuolar localized enzyme is confirmed by checking for proteinase B activity in isolated mutant vacuoles. Defective proteinase B activity segregates 2:2 in meiotic tetrads. The mutation is shown to be recessive. Mutant proteinase B activity is not only absent against the synthetic substrate. Azocoll, but also against the physiological substrate pre-chitin synthetase, cytoplasmic malate dehydrogenase and fructose-1,6-bisphosphatase. The mutant shows normal vegetative growth, a phenomenon not consistent with the idea that proteinase B might be the activating principle of chitin synthetase zymogen in vivo. Fluorescence microscopy shows normal chitin insertion. Enzymes underlying carbon-catabolite inactivation in wild-type cells (a mechanism proposed to be possibly triggered by proteinase B) such as cytoplasmic malate dehydrogenase, fructose-1,6-bisphosphatase, phosphoenolpyruvate carboxykinase and isocitrate lyase, are inactivated also in the mutant. NADP-dependent glutamate dehydrogenase, which is found to be inactivated in glucose-starved wild-type cells, proceeds normally in the mutant. Mutant cells show more than 40% reduced protein degradation under starvation conditions. Sporulating diploids, homozygous for proteinase B absence, also exhibit an approximately 40% reduced protein degradation as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene. The time of the appearance of the first ascospores of diploid cells, homozygous for proteinase B deficiency, is delayed about 50% and sporulation frequency is reduced to about the same extent as compared to homozygous wild-type diploids or diploids heterozygous for the mutant gene.
已分离出缺乏蛋白酶B活性的酵母突变体[沃尔夫，D.H.和埃曼，C.（1978年）《欧洲生物化学学会联合会快报》92，121 - 124]。其中一个突变体（HP232）已得到详细表征。通过检测分离出的突变体液泡中的蛋白酶B活性，证实了液泡定位酶的缺失。缺陷型蛋白酶B活性在减数分裂四分体中以2:2分离。该突变显示为隐性。突变体蛋白酶B活性不仅对合成底物偶氮酪蛋白无活性，而且对生理底物前几丁质合成酶、细胞质苹果酸脱氢酶和果糖-1,6-二磷酸酶也无活性。该突变体显示出正常的营养生长，这一现象与蛋白酶B可能是体内几丁质合成酶原激活因子的观点不一致。荧光显微镜检查显示几丁质插入正常。野生型细胞中碳分解代谢失活所涉及的酶（一种被认为可能由蛋白酶B触发的机制），如细胞质苹果酸脱氢酶、果糖-1,6-二磷酸酶、磷酸烯醇丙酮酸羧激酶和异柠檬酸裂解酶，在突变体中也失活。在葡萄糖饥饿的野生型细胞中被发现失活的NADP依赖性谷氨酸脱氢酶，在突变体中正常发挥作用。突变体细胞在饥饿条件下蛋白质降解减少40%以上。对于缺乏蛋白酶B的纯合二倍体孢子形成细胞，与纯合野生型二倍体或该突变基因杂合的二倍体相比，蛋白质降解也减少约40%。对于蛋白酶B缺陷纯合的二倍体细胞，第一个子囊孢子出现的时间延迟约50%，孢子形成频率与纯合野生型二倍体或该突变基因杂合的二倍体相比降低到大致相同的程度。
