The sorting and segregation mechanism of the endocytic pathway is functional in a cell-free system

A cell-free system which reconstitutes early stages of receptor-mediated endocytosis has been developed, based on detection of the association between avidin-beta-galactosidase (Av beta Gal) and biotin-transferrin (B-Tf). Initially, Av beta Gal (a fluid-phase marker) and B-Tf (receptor-bound) are internalized and delivered to a common endosomal compartment in vivo and in vitro. Subsequently, these two probes enter divergent intracellular pathways: Av beta Gal is sorted from the endosome and directed for delivery to lysosomes, whereas B-Tf is segregated away from the fluid-phase marker, remaining bound to the transferrin receptor for return to the cell surface. Using the avidin-biotin association reaction to monitor the co-localization of these two probes, we have been able to reconstruct this sorting and segregation process in a cell-free system. The in vitro reaction is time-, temperature-, and ATP-dependent, and is not affected by NH4Cl; cell-free segregation of the two probes is also sensitive to N-ethylmaleimide. As these characteristics are also properties of in vitro endocytic vesicle fusion, it is likely that the latter event is a prerequisite for the sorting and segregation process. Both the in vivo and in vitro sorting of Av beta Gal and B-Tf to their respective and distinct destinations can be followed by subcellular fractionation on Percoll gradients. Our observations provide the first evidence that the cellular mechanism to identify, sort, and sequester endocytosed material can be reconstituted in a cell-free system.