Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function
Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function
复制标题
DOI：
10.1073/pnas.1004303107
复制
发表时间：
2010-07-06
影响因子：
11.1
通讯作者：
Dudley, Jaquelin P.
中科院分区：
文献类型：
--
作者：
Byun, Hyewon;Halani, Nimita;Dudley, Jaquelin P.
小鼠乳腺肿瘤病毒（MMTV）是一种复杂的小鼠逆转录病毒，编码HIV Rev样输出蛋白Rem，来自包膜（Env）mRNA的双剪接版本。以前，Rem的N-末端98个氨基酸序列（与Env信号肽（SP）相同）和全长Rem在测量输出后功能的报告基因测定中显示出功能性。在这里，我们表明，MMTV感染的细胞或转染rem或env cDNA的细胞表达SP，这是在报告分析的活性成分。未切割的Rem部分糖基化，但在C末端内的两个糖基化位点的突变阻止Rem功能。减少Rem或Env被信号肽酶切割的突变大大降低了报告基因测定中的SP水平和功能活性，并允许未切割蛋白的积累。荧光显微镜显示，GFP标记的裂解位点突变体是不稳定的，缺乏荧光与野生型雷姆相比，这表明不正确的折叠。蛋白酶体抑制剂允许积累未切割的雷姆相对于SP和增加的报告活性，与SP的逆转位和蛋白酶体逃逸前进入核。显性负性p97 ATP酶的表达并没有改变未加工的雷姆和SP的水平，但降低了报告活性，这表明p97促进SP的retrotranslocation。我们的研究结果提供了一个例子，SP是由信号肽酶和retrotranslocated处理，使核定位和功能。
Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the N-terminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavage-site mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function.