Cell-Free Reconstitution of Multivesicular Body (MVB) Cargo Sorting

Abstract

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.