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. 2022 Apr 11;221(5):e202109084. doi: 10.1083/jcb.202109084

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© 2022 Gao et al.

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Figure 7. — HOPS mutants affect TORC1 activity. (A) Localization of VT relative to the vacuole. Wild-type (wt), vps11-1, and vps11-3 cells were transformed with the VT (Sch9^C-term-GFP-Pho8^N-term) reporter and grown in a synthetic medium at 24 or 30°C. Vacuoles were stained with FM4-64. The cells were analyzed by fluorescence microscopy and are shown as individual slices. Scale bar, 5 μm. (B) The vps11-1 allele causes changes in both VT and ET activities. Strains with the indicated genotypes were transformed with ET (FYVE-GFP-Sch9^C-term) or VT (Sch9^C-term-GFP-Pho8^N-term) reporters and grown exponentially at 24 or 30°C on SDC + all medium. To measure ET/VT activities (Hatakeyama et al., 2019), proteins were extracted and run on SDS-PAGE, and the phosphorylation levels of the ET/VT reporters were detected by immunoblotting using phospho-specific anti-Sch9-pThr⁷³⁷ antibodies. ET/VT input levels were detected with anti-GFP antibodies. Different exposures are shown to better visualize the effects on ET and VT. (C) Quantifications of the ET/VT assays in A. Significance was determined with a two-tailed Student’s t test (**, P < 0.005; *, P < 0.05). (D) Phosphorylation states of vacuolar Sch9 and endosomal Vps27. Wild-type, vps11-1, and vps11-3 were grown in synthetic complete medium. Corresponding cells extracts were run on 7.5 and 9% SDS-PAGE and probed with phosphospecific Thr⁷³⁷ Sch9 and anti-Sch9 antibodies or run on a 6% gel containing 50 μM Mn²⁺-Phos-tag and probed with anti-Vps27 antibodies. (E) Quantifications of the Sch9 Thr⁷³⁷ phosphorylation assayed in D. Error bars represent SD of three independent experiments. **, P ≤ 0.01 (Student’s t test). (F) Growth of wild-type, vps4Δ, vps11-1, vps18-1, and vps11-3 on rapamycin-containing plates. The cells were grown in synthetic medium, spotted onto plates containing SDC + all with or without 2 ng/ml rapamycin, and grown at either 24 or 30°C for 2–5 d. (G) VPS4 deletion affects vacuolar but not ET activity. Wild-type and vps4Δ cells were transformed with ET (FYVE-GFP-Sch9^C-term) or VT (Sch9^C-term-GFP-Pho8^N-term) reporters and grown exponentially at 30°C in a synthetic medium. ET/VT activities were assessed as in B. (H) Quantifications of the ET/VT assay in G. Significance was determined with a two-tailed Student’s t test (*, P ≤ 0.05). Source data are available for this figure: SourceData F7.