Department of Neurobiology and Anatomy, University of Texas Health Science Center, Houston, TX, USA.
Traffic. 2010 Jun;11(6):867-76. doi: 10.1111/j.1600-0854.2010.01053.x. Epub 2010 Feb 27.
The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.
细胞膜蛋白的数量及其在质膜上的停留时间是细胞对控制可塑性、生长和分化的信号做出反应的关键决定因素。在内化后，许多质膜蛋白的最终命运取决于它们是否被分拣到多泡体（MVB）的腔室小泡中，这是溶酶体降解的必要步骤。为了帮助阐明 MVB 分拣的机制，我们开发了一种新型的无细胞测定法，该测定法重新构建了典型膜蛋白——表皮生长因子受体的分拣，我们用它来研究其一些分子要求。所测量的分拣事件取决于细胞质、ATP、时间、温度和完整的质子梯度。Hrs 的耗竭抑制了通过在测定中包含重组 Hrs 来挽救的生化和形态学分拣测量。此外，信号转导衔接分子（STAM）的耗竭或添加突变的 ATPase 缺陷 Vps4 也抑制了分拣。该测定法重新构建了晚期内体的成熟，包括内部小泡的形成和膜蛋白的分拣，并允许对此过程进行生化研究。
