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 protei...

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Veröffentlicht in:Proceedings of the National Academy of Sciences - PNAS 2009-10, Vol.106 (41), p.17395-17400
Hauptverfasser: Tran, John H, Chen, Ching-Jen, Emr, Scott, Schekman, Randy
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Sprache:eng
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Zusammenfassung: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0909473106