Importantly, Ypq1 does not sort into puncta in the absence of rapamycin (Figure 5A bottom), indicating that the puncta are not simply due to the reduction in ATP levels

Importantly, Ypq1 does not sort into puncta in the absence of rapamycin (Figure 5A bottom), indicating that the puncta are not simply due to the reduction in ATP levels. screens to identify components of the sorting machinery required for vacuole membrane protein degradation. These screens uncovered genes that encode a ubiquitin ligase complex, components of the PtdIns 3-kinase complex, and the ESCRT machinery. We developed a novel ubiquitination system, mutant cells (Figure 1C, left). In contrast, mutants defective for Ypq1 ubiquitination and sorting (Figure 1C, right), such as and its vacuole membrane adaptor and and mutants. Cells were grown at 26C and shifted to -Lysine media at 37C for 2 hr. Arrows indicate representative Ssh4-mNeonGreen localized at pre-vacuolar compartments. (B) Ypq1-GFP was immunoprecipitated using GFP-trap resin from and cells expressing Myc-ubiquitin. Cells were grown at 26C and shifted to 37C?(non-permissive temperature) 15 min prior to lysine starvation. Cells were harvested after 2 hr lysine starvation at 37C. Both input and immunoprecipitated protein samples were analyzed using Western blot and probed with GFP and Myc antibodies. The scale bar represents 2 m. DOI: http://dx.doi.org/10.7554/eLife.26403.005 Figure 2figure supplement 2. Open in a separate window FRB-3xUb is required for rapamycin-induced Ypq1 degradation.(A) Fluorescent microscopy analysis of the RapiDeg assay. Images show cells with or without FRB-3xUb after rapamycin treatment (1 g/ml) at 30C. (B) Western blot analysis of the RapiDeg assay with or without FRB-3xUb. Whole cell lysates from both conditions were taken 0, 15, 30 and 45 min after rapamycin treatment (1 g/ml) at 30C. Blots were probed with anti-GFP and anti-G6PDH antibodies. The scale bar represents 2 m. DOI: http://dx.doi.org/10.7554/eLife.26403.006 Figure 2figure supplement 3. Open in a separate window The RapiDeg assay is highly specific.(A) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Ypq1-mCherry. Images show cells that are untreated (top), and rapamycin treated (bottom). (B) Fluorescent microscopy analysis of wild-type cells expressing Ypq1-FKBP and Ypq1-mCherry. Images show cells grown in SC-Ura (Top), and cells starved of lysine for 6 hr (Bottom). (C) Fluorescent microscopy analysis of (SNARE) and (HOPS) at non-permissive temperature (Li et al., 2015b). Since Ssh4 delivery requires these fusion machineries (Figure 2figure (S)-3-Hydroxyisobutyric acid supplement 1A), we hypothesized that the delivery of newly synthesized Ssh4 may be required and that the pre-existing vacuole membrane pool of Ssh4 may not be sufficient to ubiquitinate Ypq1 after temperature shift. We then tested whether these mutants block Ypq1 ubiquitination. As can be seen in the Figure 2figure supplement 1B, (S)-3-Hydroxyisobutyric acid these mutants block the ubiquitination of Ypq1-GFP after lysine withdrawal. Taken together, our results suggest (S)-3-Hydroxyisobutyric acid that proper delivery of Ssh4 to the vacuole membrane is required for the normal ubiquitination of Ypq1-GFP. Rapamycin-induced degradation (RapiDeg) system The Ypq1 ubiquitination defect in ESCRT mutants and endosome-vacuole fusion mutants make it difficult to study whether these components are involved in the downstream sorting steps of ubiquitinated Ypq1. To circumvent this problem, we developed a rapid and specific cargo ubiquitination system, or mutants (Li et al., 2015b), therefore, may be (S)-3-Hydroxyisobutyric acid due to indirect effects (e.g. Ssh4 sorting block). We set out to examine whether the endosome-vacuole fusion step is required for the sorting of ubiquitinated Ypq1. To do so, we used the RapiDeg system to test if Ypq1 could be sorted into the vacuole lumen in mutants that block endosome-vacuole fusion. To block endosome-vacuole fusion, we used temperature-sensitive mutants in the CORVET/HOPS complex (and mutants at the nonpermissive temperature (Figure 3A,C). These data are consistent with the Western blot results, which demonstrated that Ypq1-FKBP was degraded in the or mutants at non-permissive Tmem24 temperatures (Figure 3B,D). Open in a separate window Figure 3. Ypq1 sorting does not require membrane fusion.(A) Fluorescent microscopy analysis of Ypq1-FKBP and Vph1-mCherry in cells at non-permissive temperature (37C). The cells were grown at 26C then pretreated at 37C for 15 min. Images show before (0 min) and after (30 min) rapamycin treatment (RapiDeg). (B) Western blot analysis of Ypq1-FKBP degradation. Wild-type and cells were shifted to 37C?(15 min pretreatment) and collected at four time points (0, 15, 30, and 45.