CONFORMATIONAL MATURATION OF CFTR BUT NOT ITS MUTANT COUNTERPART (DELTA-F508) OCCURS IN THE ENDOPLASMIC-RETICULUM AND REQUIRES ATP
CONFORMATIONAL MATURATION OF CFTR BUT NOT ITS MUTANT COUNTERPART (DELTA-F508) OCCURS IN THE ENDOPLASMIC-RETICULUM AND REQUIRES ATP
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DOI：
10.1002/j.1460-2075.1994.tb06954.x
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发表时间：
1994-12-15
期刊：
影响因子：
11.4
通讯作者：
GRINSTEIN, S
中科院分区：
文献类型：
--
作者：
LUKACS, GL;MOHAMED, A;GRINSTEIN, S
通过代谢标记实验和免疫沉淀，研究了野生型（wt）和突变型（Delta F508）囊性纤维化传导跨膜调节剂（CFTR）的动力学、位置和降解抑制剂的敏感性。在生物合成过程的早期阶段，我们发现wt和Delta F508 CFTR都容易被内源性蛋白酶降解。几乎所有的Delta F508 CFTR和45-80%的wt CFTR都能快速降解，半衰期相似（t（1/2）近似于0.5 h）。无论内质网（ER）和高尔基体之间的交通是否正常，其余的wt CFTR都获得了蛋白酶抗性配置。构象转变需要代谢能，但维持抗蛋白酶wt CFTR的稳定性不需要代谢能。Delta F508 CFTR和未完全折叠的wt CFTR的细胞内降解发生在非溶酶体，前高尔基区室，这表明蛋白质水解对不同抑制剂和温度的敏感性。因此，通过免疫印迹可以在分离的内质网中检测到Delta F508 CFTR的降解产物，但在高尔基体中检测不到。总之，这些结果表明，在内质网中两种形式的wt CFTR之间存在动态平衡：一种不完全折叠、蛋白酶敏感的形式，通过atp依赖过程部分转化为一种更成熟的形式，这种形式具有蛋白酶抗性，能够离开内质网。Delta F508 CFTR无法经历这种转变，使其易于在前高尔基区室中完全和快速降解。
Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild-type (wt) and mutant (Delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and Delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all Delta F508 CFTR and 45-80% of wt CFTR were rapidly degraded with a similar half-life (t(1/2) approximate to 0.5 h). The remaining wt CFTR attained a protease-resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease-resistant wt CFTR. Intracellular degradation of Delta F508 CFTR and of incompletely folded wt CFTR occurs in a non-lysosomal, pre-Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of Delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease-sensitive form which is partially converted by an ATP-dependent process to a more mature form that is protease-resistant and capable of leaving the ER, The inability Delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre-Golgi compartment.