Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.
Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.
复制标题
中国仓鼠卵巢细胞温度敏感突变体中的溶酶体受损。
DOI：
10.1083/jcb.108.6.2211
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发表时间：
1989-06
影响因子：
7.8
通讯作者：
Draper, R K
中科院分区：
文献类型：
--
作者：
Colbaugh, P A;Stookey, M;Draper, R K
我们在这里描述的中国仓鼠卵巢细胞的突变体，表达一个条件性致死突变影响致密的溶酶体的属性。该突变体，称为V.24.1，是被选择用于对蛋白毒素具有抗性的温度敏感突变体的End 4互补组的成员（Colbaugh，P.A.，C.- Y. Kao，S.- P. Shia，M. Stookey和R. K.德雷珀。1988.体细胞分子Genet. 14：499- 507）。囊泡中存在的postnuclear上清液中制备的V.24.1细胞收获在限制性温度有50%的酸化活性减少，评估由ATP刺激的积累的染料吖啶橙子在酸性囊泡。为了研究特定群体的囊泡是否在酸化中受损，我们测量了从Percoll梯度制备的三个亚细胞组分中的酸化活性：一个含有内体和高尔基体标记物，一个含有浮力溶酶体，第三个含有致密溶酶体。致密溶酶体中的活性降低了90%，浮力溶酶体部分中的活性不受影响，并且内体-高尔基体部分中的活性轻度降低。三种溶酶体酶--β-氨基己糖苷酶、β-半乳糖苷酶和β-葡糖脑苷脂酶的活性在致密溶酶体中也降低，但在浮力溶酶体部分中几乎正常。然而，β-氨基己糖苷酶和β-葡糖脑苷脂酶活性在内体-高尔基体组分中增加了2至3倍。我们的结论是病变选择性损害致密的溶酶体，但对浮力溶酶体的性质几乎没有影响。
We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499- 507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes-- beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase-- was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta- glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.