Fig. 7. After internalization by flip, M-C₆-NBD-PC is sorted to the vacuole and must pass through the PVC. Based on analysis of membrane trafficking mutants deficient in the steps shown in the figure, we determined that M-C₆-NBD-PC must pass through the PVC in order to gain access to the lumen of the vacuole. The key result leading to this model is that class E vps mutants, which are defective in vesicular transport from the PVC to the vacuole, do not sort M-C₆-NBD-PC to the vacuole. Two models of how M-C₆-NBD-PC might gain access to the lumen of organelles in the vps pathway are illustrated — namely, internalization into the PVC by either invagination of the PVC or by a PVC-localized flippase (see Discussion for details).