Author ORCID Identifier


Date of Graduation


Document Type

Dissertation (PhD)

Program Affiliation


Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Andrew J Bean, Ph.D.

Committee Member

Peter E. Zage, M.D., Ph.D.

Committee Member

James McNew, Ph.D.

Committee Member

Guangwei Du, Ph.D.

Committee Member

M. Neal Waxham, Ph.D.


Regulating the residence time of membrane proteins (e.g. transporters, ion channels, receptors) on the cell surface can modify their response to extracellular cues and allow for cellular adaptation to environmental conditions. The fate of membrane proteins that are internalized from the plasma membrane and arrive at the limiting membrane of the late endosome/multivesicular body (MVB) is dictated by whether they remain on the limiting membrane, bud into internal MVB vesicles, or bud outwardly from the membrane. The molecular details underlying the disposition of membrane proteins that transit this pathway and the mechanisms regulating these trafficking events are unclear. We established a cell-free system that reconstitutes budding of membrane protein cargo into internal MVB vesicles and onto vesicles that bud outwardly from the MVB membrane. Both budding reactions are cytosol-dependent and supported by Saccharomyces cerevisiae (yeast) or Drosophila melanogaster (fly) cytosol, providing a system amenable to genetic manipulation. We observed that inward and outward budding are mechanistically distinct but may be linked, such that inhibition of inward budding enhances outward budding.


Endocytosis, ESCRT, multivesicular body, endosome, vesicle, cell-free assay, budding, recycling

Available for download on Thursday, April 26, 2018