B. The ER-resident protein Hmg1 is delivered to the vacuole with GFP-Snc1-PEM. The vacuole of PEP4PRB1 (WT) and pepb4∆ prb1∆ mutant cells expressing Hmg1-mCherry and overexpressing GFP-Snc1-PEM (see Panel A, left) was stained with CMAC-Arg (blue), and the cells were analyzed by live-cell microscopy. In WT cells (top), there is no intracellular Snc1-PEM and the red-labeled ER is distinguished from the blue vacuole. In contrast, in ~75% pep4∆ prb1∆ mutant cells (bottom), which are defective in vacuolar proteolysis, Hmg1 (red) co-localizes with Snc1-PEM (green) in the vacuole (blue). Shown from left to right: DIC, GFP, mCherry, blue, merge (white shows merge of the three colors), % cells with intracellular Snc1-PEM, % cells in which Hmg1 with Snc1-PEM co-localize, and % co-localization of the two proteins with the vacuolar dye (38 cells were analyzed for each strain). 