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. 2024 Mar 27;223(6):e202305038. doi: 10.1083/jcb.202305038

Figure S1.

Figure S1.

Gyp7 does not affect Ypt7 function in autophagy or vCLAMP formation. (A) Localization of Gyp7 in selected deletion mutants. Gyp7 was tagged with mNeonGreen in wild-type (wt), vps21Δ, ypt52Δ, ypt10Δ, ypt53Δ, vps9Δ muk1Δ, snx4Δ, vps5Δ, vps35Δ, and vps38Δ cells. Vacuolar membranes were stained with FM4-64. Cells were imaged by fluorescence microscopy and individual slices are shown. Dashed lines indicate yeast cell boundaries. Scale bar, 2 μm. (B) Localization of Atg8 upon deletion or overexpression of Gyp7. Gyp7 was either deleted or expressed from the TEF1 promoter in cells encoding the autophagy-specific marker protein mCherry-Atg8. Cells were grown in SDC+all and then shifted to SD-N for 1 h (see Materials and methods). Cells were imaged by fluorescence microscopy and individual slices are shown. Dashed lines indicate yeast cell boundaries. Scale bar, 2 μm. (C) Quantification of Atg8 dots per cell in B during growth. Cells (n ≥ 150) from three independent experiments were quantified in Fiji. Bar graphs represent the averages from three experiments and dots represent the mean of each experiment. P value ns, **<0.01, using ANOVA one-way test. (D) Quantification of Atg8 dots per cell in B during N-starvation. Quantification was performed as in C. P value ns, using ANOVA one-way test. (E) Formation of vCLAMPs upon deletion or overexpression of Gyp7. The vCLAMP-forming protein mCherry-Vps39 was expressed from the TEF1 promoter in cells encoding Gyp7-mNeonGreen, gyp7Δ, and TEF1pr-GYP7 cells. Mitochondria were stained with DAPI (see Materials and methods). Cells were imaged by fluorescence microscopy and individual slices are shown. Dashed lines indicate yeast cell boundaries. Scale bar, 2 μm. (F) Quantification of E. Colocalization of mCherry-Vps39 enrichments and DAPI-stained mitochondria was defined as vCLAMP. Cells with ≥1 vCLAMP were counted as vCLAMP-positive cells. Cells (n ≥ 150) from three independent experiments were quantified in Fiji. Bar graphs represent the averages from three experiments and dots represent the mean of each experiment. P value ns, using ANOVA one-way test.