A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature
A common temperature-sensitive allelic form of human tyrosinase is retained in the endoplasmic reticulum at the nonpermissive temperature
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DOI：
10.1074/jbc.275.16.12281
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发表时间：
2000-04-21
影响因子：
4.8
通讯作者：
Marks, MS
中科院分区：
文献类型：
--
作者：
Berson, JF;Frank, DW;Marks, MS
眼皮肤白化病1型TS是由突变引起的，使黑素细胞特异性酶酪氨酸酶温度敏感（ts）;该酶在37摄氏度生长的细胞中无活性，但在31摄氏度生长的细胞中显示出完全活性。要区分是否TS表型的常见的R402 Q变体的人酪氨酸酶是由于改变酶活性或错误折叠和细胞内运输的缺陷，我们分析了其定位和处理在瞬时转染HeLa细胞。R402 Q酪氨酸酶在37 ℃时在内质网（ER）中积累，但在31 ℃时离开ER并在细胞内体结构中积累。R402 Q变体不能离开ER通过在37 ° C下不能获得糖苷内切酶H抗性来证实，并且不能仅通过增强的蛋白酶体介导的降解来解释。ER在37 ℃的滞留是由R402 Q酪氨酸酶的内腔结构域介导的，不依赖于与膜的束缚，并且是不可逆的。最后，酪氨酸酶的野生型等位基因形式在瞬时转染的HeLa细胞中是部分ts。数据显示，在非黑素细胞中表达的人酪氨酸酶折叠并低效地离开ER，并且R402 Q酪氨酸酶夸大了这种缺陷，导致在生理温度下不能离开ER。
Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37 degrees C but displays full activity in cells grown at 31 degrees C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37 degrees C but exits the ER and accumulates in endosomal structures in cells grown at 31 degrees C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37 degrees C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37 degrees C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.