Cell-free reconstitution of multivesicular body formation and receptor sorting.
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.
Sun
Wei
W
Department of Neurobiology and Anatomy, University of Texas Health Science Center, Houston, TX, USA.
Vida
Thomas A
TA
Sirisaengtaksin
Natalie
N
Merrill
Samuel A
SA
Hanson
Phyllis I
PI
Bean
Andrew J
AJ
D000074183:ATPases Associated with Diverse Cellular Activities; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004705:Endocytosis; D056827:Endosomal Sorting Complexes Required for Transport / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008854:Microscopy, Electron / Q000379:methods; D008954:Models, Biological; D010750:Phosphoproteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D025262:Vacuolar Proton-Translocating ATPases
D048868:Adaptor Proteins, Signal Transducing; D056827:Endosomal Sorting Complexes Required for Transport; D010750:Phosphoproteins; C109775:STAM protein, human; D000251:Adenosine Triphosphatases; D025262:Vacuolar Proton-Translocating ATPases; D000074183:ATPases Associated with Diverse Cellular Activities; C490305:VPS4A protein, human