The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein.
The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein.
复制标题
Charcot Marie牙齿疾病蛋白LITAF是一种锌结合的单位膜蛋白。
DOI：
10.1042/bcj20160657
复制
发表时间：
2016-11-01
期刊：
影响因子：
--
通讯作者：
Woodman PG
中科院分区：
文献类型：
--
作者：
Qin W;Wunderley L;Barrett AL;High S;Woodman PG
LITAF（LPS诱导的TNF活化因子）是一种内体相关的整合膜蛋白，对于多泡体分选很重要。LITAF中的几种突变导致常染色体显性Charcot玛丽牙病1C型。这些突变映射到高度保守的C-末端区域，称为LITAF结构域，其包括22个残基的疏水序列和侧翼富含半胱氨酸的区域，其含有在锌指中发现的肽基序。虽然LITAF结构域被认为是负责膜整合，LITAF的膜拓扑结构尚未建立。在这里，我们研究了LITAF是否是一个尾部锚定（TA）跨膜蛋白或单调的膜蛋白。当在体外翻译时，与真正的TA蛋白Sec 61 β相比，LITAF很难整合到ER衍生的微粒体中。此外，N-连接的糖基化报告基因的引入表明LITAF的N-末端和C-末端结构域都没有易位到ER腔中。含有C-末端糖基化位点的LITAF构建体在细胞中的表达证实了LITAF不是细胞中的TA蛋白。最后，基于免疫荧光的潜伏期测定显示LITAF的N-和C-末端都暴露于细胞质。重组LITAF含有1 mol/mol锌，而预测的锌结合残基的突变破坏LITAF膜缔合。 因此，我们得出结论，LITAF是一个单调的膜蛋白，其膜整合是稳定的锌指。相关的人类蛋白CDIP 1（细胞死亡涉及p53靶点1）显示出相同的膜拓扑结构，表明这种膜整合模式在LITAF家族蛋白中是保守的。
LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61β, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.