Mittenbühler K, Holzer H
Biochemisches Institut, Universität Freiburg, Federal Republic of Germany.
Arch Microbiol. 1991;155(3):217-20. doi: 10.1007/BF00252203.
The biosynthesis and processing of the vacuolar (lysosomal) acid trehalase (molecular mass about 220 kDa) was followed in vivo using mutants conditionally defective in the secretory pathway. A precursor of 41 kDa was found in sec61 mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum. Endoglycosidase H and N-glycosidase F treatment of purified acid trehalase in vitro resulted in a 41 kDa band, indicating that the precursor form found in sec61 mutant cells corresponds to the carbohydrate-free form of the enzyme. sec18 mutant cells, blocked in the delivery of secretory proteins from the endoplasmic reticulum to the Golgi body accumulate a form with a molecular mass of 76 kDa which probably corresponds to a partially glycosylated precursor of the mature acid trehalase. This precursor partially disappears in favour of the appearance of a higher molecular weight component of 180 kDa in sec7 mutants which are blocked in the delivery step of secretory proteins from the Golgi body to the vacuole. In wild-type cells the fully glycosylated mature form of acid trehalase of about 220 kDa was observed accompanied by some 180 kDa and 76 kDa material.
利用在分泌途径中存在条件缺陷的突变体，在体内追踪液泡（溶酶体）酸性海藻糖酶（分子量约220 kDa）的生物合成和加工过程。在分泌蛋白前体向内质网腔转运存在缺陷的sec61突变体细胞中，发现了一种41 kDa的前体。用内切糖苷酶H和N-糖苷酶F在体外处理纯化的酸性海藻糖酶，产生了一条41 kDa的条带，这表明在sec61突变体细胞中发现的前体形式对应于该酶的无糖基化形式。sec18突变体细胞在分泌蛋白从内质网向高尔基体的转运过程中受阻，积累了一种分子量为76 kDa的形式，这可能对应于成熟酸性海藻糖酶的部分糖基化前体。在sec7突变体中，这种前体部分消失，取而代之的是一种分子量为180 kDa的更高分子量成分的出现，sec7突变体在分泌蛋白从高尔基体向液泡的转运步骤中受阻。在野生型细胞中，观察到约220 kDa的完全糖基化成熟形式的酸性海藻糖酶，同时伴有一些180 kDa和76 kDa的物质。
