Generation and properties of the MKO strain. (A) Schematic representation of the MKO strategy. The loxP–Cre system allows disruption of up to five genes using different markers, and efficient removal of markers by Cre expression. Multiple rounds of deletion and marker rescue yielded the MKO strain. See Materials and methods for detailed information. (B) Growth curve of the MKO strain compared with wild-type (WT) and atg1Δ strains. WT, atg1Δ, and the MKO strains were grown overnight in YPD and diluted to OD₆₀₀ = 0.1. Aliquots were removed for OD₆₀₀ readings every hour for 13 h. (C) Morphology of the MKO strain. WT and atg18Δ strains and the MKO strain with or without a plasmid expressing Atg18 (pATG18(415)) were grown in YPD to mid-log phase and observed by microscopy. The MKO strain had an enlarged vacuole similar to the atg18Δ strain; once transformed with Atg18, the vacuole size became the same as that in the wild type. Bar, 2 μm. (D) The MKO strain expressing Atg6 is not defective for the carboxypeptidase Y, MVB, or alkaline phosphatase pathways, and was assayed by pulse-chase experiments for Prc1, Cps1 (not depicted), and Pho8 processing. The atg6Δ and MKO strains transformed with a plasmid expressing Atg6 (pATG6(414)) or the empty pRS414 vector were subjected to a radioactive label/nonradioactive chase and triple-immunoprecipitated as described in Materials and methods, then analyzed by 8% SDS-PAGE. Carboxypeptidase Y (Prc1) transits through the ER in a precursor (p1) form, acquires its p2 form in the Golgi complex, and, via the late endosome–MVB pathway, is delivered to the vacuole and converted to its mature (m) form. Vacuolar alkaline phosphatase (Pho8) is transported through the ER to the Golgi in a precursor (p) form but bypasses the endosome and is proteolytically activated to its mature (m) form in the vacuole. Atg6 is not required for the alkaline phosphatase pathway; however, for consistency, cells expressing Atg6 were used throughout the experiment.