Gireud Monica, Sirisaengtaksin Natalie, Tsunoda Susan, Bean Andrew J
Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, TX, 77030, USA.
Methods Mol Biol. 2015;1270:115-24. doi: 10.1007/978-1-4939-2309-0_9.
The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded. Following internalization and movement through the endocytic pathway en route to lysosomes, membrane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting membrane and containing internal vesicles. The fate of an internalized membrane protein depends on whether it buds outwardly from the endosomal membrane, promoting recycling and continued signaling, or is internalized into internal MVB vesicles and is ultimately degraded upon MVB-lysosome fusion. The molecular machinery that regulates the separation of membrane proteins destined for degradation from those resulting in surface expression is not well understood.To elucidate the molecular mechanisms that underlie membrane protein sorting, we have reconstituted an endosomal sorting event under cell-free conditions. We took advantage of the itinerary of a prototypical membrane protein, the epidermal growth factor receptor (EGFR) and designed a biochemical monitor for cargo movement into internal MVB vesicles that is generally modifiable for other membrane proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle formation. We have determined that MVB sorting is dependent on cytosolic components, adenosine triphosphate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation of late endosomes and allows the morphological and biochemical examination of vesicle formation and membrane protein sorting.
细胞表面定位的膜蛋白的信号传导活性主要发生在这些蛋白位于质膜上时，但在某些情况下，直到蛋白被降解才终止。内化并通过内吞途径向溶酶体移动后，膜蛋白会穿过一种称为多囊泡体（MVB）的晚期内体细胞器。MVB是由内体的限制膜内陷形成的，形成一种具有限制膜并含有内部囊泡的细胞器。内化膜蛋白的命运取决于它是从内体膜向外出芽，促进再循环和持续信号传导，还是被内化到内部MVB囊泡中，并最终在MVB与溶酶体融合时被降解。调节注定要降解的膜蛋白与导致表面表达的膜蛋白分离的分子机制尚不清楚。为了阐明膜蛋白分选的分子机制，我们在无细胞条件下重建了内体分选事件。我们利用了一种典型膜蛋白——表皮生长因子受体（EGFR）的行程，并设计了一种生化监测器，用于监测货物进入内部MVB囊泡的移动，这种监测器通常可针对其他膜蛋白进行修改。由于尚不清楚内部囊泡形成与货物分选之间的关系，因此使用透射电子显微镜（TEM）进行形态学检查可以单独监测囊泡形成。我们已经确定MVB分选依赖于胞质成分、三磷酸腺苷（ATP）、时间、温度和完整的质子梯度。该测定法重建了晚期内体的成熟过程，并允许对囊泡形成和膜蛋白分选进行形态学和生化检查。
