Timchak L M, Kruse F, Marnell M H, Draper R K
J Biol Chem. 1986 Oct 25;261(30):14154-9.
We previously described the isolation and preliminary characterization of a Chinese hamster ovary cell mutant, termed G.7.1, that carried a temperature-sensitive, conditional-lethal lesion affecting the acidification of vesicles in crude cellular extracts (Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D. K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907-1916). In the present report, we have separated lysosomal vesicles from more buoyant nonlysosomal vesicles by centrifuging cell extracts with Percoll and correlated the acidification defect with nonlysosomal vesicles, including endosomes, but not with secondary lysosomes. Moreover, the acidification of nonlysosomal vesicles prepared from mutant cells grown at the permissive temperature was more sensitive to thermal inactivation than similar vesicles from parental cells, implying that a heat-sensitive component is a normal resident of nonlysosomal vesicles in the mutant. This heat-sensitive component is apparently not associated with lysosomes, or if it is, it does not inhibit lysosomal acidification at the nonpermissive temperature. We also found that the transferrin-mediated uptake of iron is inhibited by 50% in the mutant cells at the nonpermissive temperature and that the inhibition cannot be accounted for by reduced binding or internalization of transferrin.
我们之前描述过一种中国仓鼠卵巢细胞突变体的分离及初步特征，该突变体称为G.7.1，它带有一个温度敏感的条件致死损伤，影响粗细胞提取物中囊泡的酸化（Marnell, M. H., Mathis, L. S., Stookey, M., Shia, S.-P., Stone, D. K., and Draper, R. K. (1984) J. Cell Biol. 99, 1907 - 1916）。在本报告中，我们通过用Percoll离心细胞提取物，将溶酶体囊泡与浮力更大的非溶酶体囊泡分离，并将酸化缺陷与非溶酶体囊泡（包括内体）相关联，而与次级溶酶体无关。此外，在允许温度下生长的突变体细胞制备的非溶酶体囊泡的酸化比亲代细胞类似囊泡对热失活更敏感，这意味着一种热敏感成分是突变体中非溶酶体囊泡的正常驻留成分。这种热敏感成分显然与溶酶体无关，或者即便有关，在非允许温度下它也不会抑制溶酶体酸化。我们还发现，在非允许温度下，突变体细胞中铁的转铁蛋白介导摄取被抑制了50%，并且这种抑制不能用转铁蛋白结合或内化减少来解释。
