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Cell-free reconstitution of multivesicular body formation and receptor sorting.
- Source :
-
Traffic (Copenhagen, Denmark) [Traffic] 2010 Jun; Vol. 11 (6), pp. 867-76. Date of Electronic Publication: 2010 Feb 27. - Publication Year :
- 2010
-
Abstract
- The number of surface membrane proteins and their residence time on the plasma membrane are critical determinants of cellular responses to cues that can control plasticity, growth and differentiation. After internalization, the ultimate fate of many plasma membrane proteins is dependent on whether they are sorted for internalization into the lumenal vesicles of multivesicular bodies (MVBs), an obligate step prior to lysosomal degradation. To help to elucidate the mechanisms underlying MVB sorting, we have developed a novel cell-free assay that reconstitutes the sorting of a prototypical membrane protein, the epidermal growth factor receptor, with which we have probed some of its molecular requirements. The sorting event measured is dependent on cytosol, ATP, time, temperature and an intact proton gradient. Depletion of Hrs inhibited biochemical and morphological measures of sorting that were rescued by inclusion of recombinant Hrs in the assay. Moreover, depletion of signal-transducing adaptor molecule (STAM), or addition of mutated ATPase-deficient Vps4, also inhibited sorting. This assay reconstitutes the maturation of late endosomes, including the formation of internal vesicles and the sorting of a membrane protein, and allows biochemical investigation of this process.
- Subjects :
- ATPases Associated with Diverse Cellular Activities
Animals
Brain metabolism
Cell Membrane metabolism
Endocytosis
Endosomes metabolism
HeLa Cells
Humans
Lysosomes metabolism
Microscopy, Electron methods
Models, Biological
Protein Structure, Tertiary
Rats
Vacuolar Proton-Translocating ATPases
Adaptor Proteins, Signal Transducing metabolism
Adenosine Triphosphatases metabolism
Cell-Free System
Endosomal Sorting Complexes Required for Transport metabolism
Phosphoproteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1600-0854
- Volume :
- 11
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Traffic (Copenhagen, Denmark)
- Publication Type :
- Academic Journal
- Accession number :
- 20214752
- Full Text :
- https://doi.org/10.1111/j.1600-0854.2010.01053.x