Biomedical Engineering Reference
In-Depth Information
Chapter 9
Cell-Free Reconstitution of Multivesicular
Body (MVB) Cargo Sorting
Monica Gireud , Natalie Sirisaengtaksin , Susan Tsunoda ,
and Andrew J. Bean
Abstract
The signaling activity of cell surface localized membrane proteins occurs primarily while these proteins are
located on the plasma membrane but is, in some cases, not terminated until the proteins are degraded.
Following internalization and movement through the endocytic pathway en route to lysosomes, mem-
brane proteins transit a late endosomal organelle called the multivesicular body (MVB). MVBs are formed
by invagination of the limiting membrane of endosomes, resulting in an organelle possessing a limiting
membrane and containing internal vesicles. The fate of an internalized membrane protein depends on
whether it buds outwardly from the endosomal membrane, promoting recycling and continued signaling,
or is internalized into internal MVB vesicles and is ultimately degraded upon MVB-lysosome fusion. The
molecular machinery that regulates the separation of membrane proteins destined for degradation from
those resulting in surface expression is not well understood.
To elucidate the molecular mechanisms that underlie membrane protein sorting, we have reconsti-
tuted an endosomal sorting event under cell-free conditions. We took advantage of the itinerary of a pro-
totypical membrane protein, the epidermal growth factor receptor (EGFR) and designed a biochemical
monitor for cargo movement into internal MVB vesicles that is generally modifi able for other membrane
proteins. Since is it not known how internal vesicle formation is related to cargo sorting, morphological
examination using transmission electron microscopy (TEM) allows separate monitoring of vesicle forma-
tion. We have determined that MVB sorting is dependent on cytosolic components, adenosine triphos-
phate (ATP), time, temperature, and an intact proton gradient. This assay reconstitutes the maturation
of late endosomes and allows the morphological and biochemical examination of vesicle formation and
membrane protein sorting.
Key words
Multivesicular body, Membrane traffi cking, Endocytic, Endosome, Sorting, Cell-free
assay
1
Introduction
Endocytosis is required for the uptake of essential nutrients
from the extracellular environment and to remove membrane
proteins from the plasma membrane [
1
]. The endocytic path-
way is separated into stages based on cargo movement through
