Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.
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DOI：
10.1091/mbc.e10-09-0742
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发表时间：
2010-12
影响因子：
3.3
通讯作者：
Hegde RS
中科院分区：
文献类型：
--
作者：
Emerman AB;Zhang ZR;Chakrabarti O;Hegde RS
一种用于检测蛋白质的次要交替定位群体的选择性标记方法被用于研究与疾病相关的朊病毒蛋白的跨膜形式。分析揭示了跨膜朊病毒蛋白代谢的关键特征，以及人类致病突变体改变这种代谢的一种方式。蛋白质通常以一种以上的形式产生，有时存在于与主要物种不同的细胞区室中的替代版本。哺乳动物朊病毒蛋白（PrP），一种细胞表面GPI锚定蛋白，是一个特别值得注意的例子，其中次要的胞质和跨膜形式与疾病发病机制有关。为了研究这些次要物种，我们使用了一种选择性标记策略，其中将生物素化酶的空间限制性表达与同源受体序列的不对称工程结合到PrP中。使用这种方法，我们可以表明，即使是野生型PrP产生少量的CtmPrP跨膜形式。CtmPrP的选择性检测使我们能够揭示其N-末端加工，长半衰期，在细胞内和细胞表面的位置，并最终在溶酶体中的降解。令人惊讶的是，PrP中的一些人类致病突变体选择性地稳定了CtmPrP，揭示了可能导致疾病的先前未预料到的CtmPrP上调机制。因此，时空标签揭示了新的方面的正常和突变的PrP代谢，并应容易适用于其他蛋白质的次要拓扑异构体的分析。
A selective tagging method for detecting minor alternatively-localized populations of a protein is used to study a disease-associated transmembrane form of prion protein. The analysis reveals key features of transmembrane prion protein metabolism and one way this is altered by human disease-causing mutants. Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the CtmPrP transmembrane form. Selective detection of CtmPrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized CtmPrP, revealing a previously unanticipated mechanism of CtmPrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.