Cargo sorting into multivesicular bodies in vitro Journal Article
| Authors: | Tran, J. H.; Chen, C. J.; Emr, S.; Schekman, R. |
| Article Title: | Cargo sorting into multivesicular bodies in vitro |
| Abstract: | 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. |
| Keywords: | controlled study; unclassified drug; nonhuman; protein domain; gene; in vitro study; bacterial protein; ubiquitination; escherichia coli; yeast; adenosine triphosphate; lysine; cytosol; internalization; biotinylation; biotin; ligase; trypsin; endosome; multivesicular bodies; reconstitution; carboxypeptidase; carboxypeptidase s; bacterium mutant; species; vacuolar protein sorting 27 gene; vacuolar protein sorting 4p gene |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 106 |
| Issue: | 41 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 2009-10-13 |
| Start Page: | 17395 |
| End Page: | 17400 |
| Language: | English |
| DOI: | 10.1073/pnas.0909473106 |
| PROVIDER: | scopus |
| PMCID: | PMC2762664 |
| PUBMED: | 19805166 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 2" - "Export Date: 30 November 2010" - "CODEN: PNASA" - "Source: Scopus" |
