Cargo sorting into multivesicular bodies in vitro.

Genetic studies have identified a number of proteins required for the internalization of biosynthetic and endocytic cargo proteins transported to the multivesicular body (MVB). We have developed a cell-free reaction that recapitulates the internalization of a yeast biosynthetic membrane cargo protein, carboxypeptidase S (CPS), into the interior of an endosome. A recombinant form of CPS containing a biotinylation site from an Escherichia coli protein is accumulated in a vps27 yeast mutant blocked in the MVB internalization event. Endosomes isolated from the vps27 mutant are exposed to E. coli biotin ligase, which acts on only those CPS molecules with a cytosol-exposed N-terminal domain. Internalization of biotin-tagged CPS is measured by the detection of trypsin-inaccessible, membrane-protected species. Biotinylated CPS internalization requires ATP and functional forms of Vps27p and Vps4p and depends on the availability of an exposed lysine residue critical for CPS ubiquitylation.