Cell-free Reconstitution of Transport from the trans-Golgi Network to the Late Endosome/Prevacuolar Compartment

Abstract

Vesicle-mediated transport between the trans-Golgi network (TGN) and the late endosome/prevacuolar compartment (PVC) is an essential step in lysosomal/vacuolar biogenesis. In addition, localization of integral membrane proteins to the TGN requires continual cycles of vesicular transport between the TGN and endosomal compartments. Genetic and biochemical analyses in yeast have identified a variety of proteins required for TGN-to-PVC transport. However, the precise mechanisms of vesicle formation, transport, and fusion have not been fully elucidated. To study the steps of TGN-to-PVC transport in mechanistic detail, we have developed a cell-free assay to monitor delivery of the processing protease Kex2p from the TGN to PVC compartments containing a Kex2p substrate. Transport is time-, temperature-, and ATP-dependent and requires the t-SNARE Pep12p. Moreover, cell-free delivery of Kex2p to the PVC results in the co-integration of Kex2p into PVC membranes containing the Kex2p substrate as determined by co-immunoisolation of Kex2p and the substrate using antibody against the Kex2p cytosolic tail. This work represents the first cell-free reconstitution and biochemical analysis of the essential vacuolar/lysosomal sorting step TGN to late endosome transport.