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. 2022 Jun 29;221(8):e202103048. doi: 10.1083/jcb.202103048

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© 2022 Bisinski et al.

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Figure S5. — Cvm1-mediated contacts are induced by a decrease in complex sphingolipids, but protein levels of Cvm1 are not altered. Related to Fig. 8. (A) Cvm1 accumulates at the NVJ upon depletion of sphingolipids. Fluorescence microscopy analysis of the localization of GFP-Cvm1 under the control of the endogenous promoter, Nvj1-mKate2 as a NVJ marker, and CMAC staining as a vacuolar marker. Cells were grown in rich media (YPAD) and incubated with 250 nM AbA for 30 min where indicated. Under normal growth conditions, Cvm1 is accumulated in the NVJ in some cells and not in others. After incubation with AbA, the accumulations of Cvm1 at the NVJ become more pronounced and happen in more cells. Scale bar represents 2 μm. BF = Brightfield. (B) Cvm1 accumulations at the PerVale are more frequent upon decreasing sphingolipid levels. Fluorescence microscopy analysis of the localization of GFP-Cvm1 under the control of the endogenous promoter, Pex3-mKate2 as a peroxisomal marker, and CMAC staining as a vacuolar marker. Cells were grown in YPAD and incubated with 5 μM AbA for 60 min where indicated. Scale bar represents 2 μm. (C) Cvm1 accumulations at the vCLAMP are more frequent upon decreasing sphingolipid levels. Fluorescence microscopy analysis of the localization of GFP-Cvm1 under the control of the endogenous promoter, Shm1-mKate2 as a mitochondrial marker, and CMAC staining as a vacuolar marker. Cells were grown in YPD and incubated with 5 μM AbA for 60 min where indicated. The yellow arrowheads indicate accumulations of Cvm1 on the vacuole membrane that co-localize with the mitochondrial network. Scale bar represents 2 μm. (D) Cvm1 protein levels are not affected by myriocin treatment. Whole-cell lysates of a strain containing GFP-Cvm1 under the control of its endogenous promoter were normalized by total protein amount and analyzed by SDS-PAGE and Western blot. The levels of GFP-Cvm1 do not change significantly throughout the treatment with the drug. Pgk1 was used as a loading control. Source data are available for this figure: SourceData FS5.