Cargo sorting into multivesicular bodies in vitro Academic Article uri icon


MeSH Major

  • Carboxypeptidases


  • 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.

publication date

  • October 13, 2009



  • Academic Article



  • eng

PubMed Central ID

  • PMC2762664

Digital Object Identifier (DOI)

  • 10.1073/pnas.0909473106

PubMed ID

  • 19805166

Additional Document Info

start page

  • 17395

end page

  • 400


  • 106


  • 41