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After proteins are synthesized, the cell's transport machinery shuttles them to their final site of action. Robert Fuller and his group at the University of Michigan have long been interested in a particular hub in the yeast cell, the trans-Golgi network (TGN). In a recent article in the Journal of Biological Chemistry, they describe a cell-free assay that they developed to hone in on the intersection between TGN and the late endosomal prevacuolar compartment (PVC), where proteins are sorted for recycling or degradation (Blanchette et al., 2004). Their goal was to biochemically identify the players in TGN to PVC trafficking.

A TGN membrane, bearing a TGN-specific protease, was mixed with a PVC membrane harboring a three component fusion protein consisting of (i) a protein whose role in TGN-to-PVC transfer was to be tested (ii) a reporter enzyme and (iii) a protease cleavage site followed by an epitope tag. If the protein of interest was involved in TGN-to-PVC membrane trafficking, it translocated the protease and the tag was subsequently cleaved. The efficacy of the transfer could then easily be quantified with the reporter enzyme. If the protein failed to transfer the protease, the fusion protein remained uncleaved and was removed by immunoprecipitation with the tag. Using this assay Fuller proved the essential role of the docking protein Pep12p for TGN-to-PVC trafficking and will now investigate other candidates such as clathrin or dynamin homologs.

“The advantage of this assay,” Fuller says, “is that it is rapid, does not involve radioactivity or gels and is very quantitative.” Fuller hopes to reconstruct the full mechanism in vitro, from TGN-vesicle budding to targeting and fusion with the late endosome–PVC vesicles. These findings will aid understanding of how the cell distinguishes between proteins targeted for recycling or degradation and those destined for secretion or localization elsewhere in the cell.