In vitro assays differentially recapitulate protein export from the trans-Golgi network

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In vitro assays have been broadly applied to reconstitute traYcking events along the biosynthetic and endocytic pathways of eukaryotic cells [1]. Cell-based assays employ mild detergents or mechanical means to perforate the plasma membrane and release cytosolic components while maintaining the integrity of intracellular membrane compartments (Supplementary Fig. 1). In contrast, cell-free assays are performed using isolated membrane fractions enriched in a particular organelle. Cell-based assays can allow analysis of multiple steps along a traYcking pathway, while cell-free assays are generally designed to reconstitute an individual membrane Wssion or fusion step. Both cellbased and cell-free assays have proven useful for dissecting the roles of various components in intracellular traYcking events. In many instances, these assays have been utilized to dissect the requirements for polarized biosynthetic sorting of cargo molecules from the trans-Golgi network (TGN)1[2–5]. Interestingly, segregation of apical and basolateral marker proteins into distinct transport carriers that bud from the TGN appears to occur even in nonpolarized cells, enabling the use of nondiVerentiated cell types for such assays [6–8]. However, the extent to which these assays recapitulate in vivo sorting and regulation of membrane traYc has not been systematically examined. Here we have compared cell-based and a cell-free in vitro assays for their ability to reconstitute polarized protein sorting from the TGN. We previously found that eYcient apical delivery of the marker protein inXuenza hemagglutinin (HA) in polarized Madin–Darby canine kidney (MDCK) cells requires TGN acidiWcation [9]. In particular,