| 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 biotinylatable sequence from E. coli is accumulated in a vps27 yeast mutant blocked in the MVB internalization event. Endosomes isolated by gentle rupture of vps27 mutant spheroplasts are exposed to E. coli biotin ligase, which acts on only those CPS molecules with a cytoplasmically exposed N-terminal domain. Internalization of biotin-tagged CPS is measured by the detection of trypsin inaccessible, membrane-protected species using SDS-PAGE and immunoblot analysis with anti-biotin antibody. 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. |
