Fig. 4.

Ume6 negatively regulates autophagy. (A) Autophagy as measured by the Pho8Δ60 assay is increased in ume6Δ cells. Wild-type (YCB193, SEY6210), atg1Δ (YCB194), and ume6Δ (YCB197) cells were grown in SMD medium and then starved for 0, 0.5, 1, 2, 3, and 4 h. The Pho8Δ60 activity was measured as described in Materials and Methods and normalized to the activity of the wild-type cells, which was set to 100%. Error bars indicate the SEM of three independent experiments. (B) Autophagosome size is increased in ume6Δ cells. Wild-type (FRY143, SEY6210) and ume6Δ (YCB234) strains with deletions of VPS4 and PEP4 to eliminate vesicles generated from the multivesicular body pathway and the breakdown of autophagic bodies, respectively, were grown in rich medium and starved in SD-N for 2 h. Samples were collected, prepared, and examined by TEM as described in Materials and Methods. The radius of each autophagosome was determined as described in Materials and Methods. The error represents the SEM for >400 autophagic bodies. (C) Representative TEM images of the cells in B. (Scale bars: 500 nm.)