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.
Tran
John H
JH
Department of Molecular and Cell Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA. tranj1@mskcc.org
Chen
Ching-Jen
CJ
Emr
Scott
S
Schekman
Randy
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D019921:Biotinylation; D002268:Carboxypeptidases / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D056827:Endosomal Sorting Complexes Required for Transport; D011992:Endosomes / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D050506:Mutant Chimeric Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D050506:Mutant Chimeric Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C119627:VPS27 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D000255:Adenosine Triphosphate; D002268:Carboxypeptidases