Journal: bioRxiv

Article Title: Yeast TLDc domain-containing proteins control assembly and subcellular localization of the V-ATPase

doi: 10.1101/2023.08.21.554079

Figure Lengend Snippet: (A) Comparison of the AlphaFold model generated for Rtc5 with the available structure of Oxr1 (PDB: 7FDE). These two TLDc domain-containing proteins are in good structural alignment (root mean square deviation (RMSD) of 3.509Å). (B and C) Same experiment as and , with switched heavy and light labeling. Vacuoles from cells lacking RTC5 or OXR1 contain more assembled V-ATPase. SILAC-based vacuole proteomics of cells lacking either OXR1 (B) or RTC5 (C) compared with the wt strain. Log10 of the detected protein intensities are plotted against Log2 of the heavy/light SILAC ratios. Significant outliers are color coded in red (P < 1e−14), orange (P < 0.0001), or dark blue (P < 0.05); other identified proteins are shown in light blue. The vacuoles of both mutant strains show increased amounts of peripheral domain V-ATPase subunits compared to the wt strain, but the effect is stronger for cells lacking OXR1 than for cells lacking RTC5 . For comparison, subunit Vma6 of the membrane-embedded domain of the V-ATPase is not significantly enriched. In addition, vacuoles of cells lacking OXR1 contain increased amounts of the Golgi-localized isoform of the membrane-embedded subunit a, Stv1. For the the individual dots to be clear, the range chosen for the X axis in panel B excludes the dot representing de protein Cwp1, which showed a Log2(normalized H/L ratio) of -2,367955 and an Log10(intensity) of 6.705487389. (D and E) Vacuoles from cells overexpressing Rtc5 or Oxr1 show no significant changes in V-ATPase assembly. SILAC-based vacuole proteomics of cells overexpressing either Oxr1 (D) or Rtc5 (E) compared with the wt strain. Log10 of the detected protein intensities are plotted against Log2 of the heavy/light SILAC ratios. Significant outliers are color coded in red (P < 1e−14), orange (P < 0.0001), or dark blue (P < 0.05); other identified proteins are shown in light blue. V-ATPase subunits are labeled and shown as green dots, and show no significant changes in the mutant strains compared to the wt. So that the individual dots are clearly visible, the range chosen for the X axis excludes the dot representing Rtc5. This protein showed a Log2(normalized H/L ratio) of 4.109945 and an Log10(intensity) 9.433689846.

Article Snippet: Rtc5 is a protein of unknown function, predicted to contain a TLDc domain ( T re2/Bub2/Cdc16, L ysM, d omain c atalytic, Pfam PF07534, InterPro IPR006571, Prosite PS51886, SMART SM00584).

Techniques: Comparison, Generated, Labeling, Multiplex sample analysis, Mutagenesis, Membrane

Journal: bioRxiv

Article Title: Yeast TLDc domain-containing proteins control assembly and subcellular localization of the V-ATPase

doi: 10.1101/2023.08.21.554079

Figure Lengend Snippet: (A - C) Stv1-mNeonGreen partially re-localizes from the late-Golgi to the vacuole in the absence of Oxr1. Fluorescence microscopy analysis of the localization of Stv1 in the absence of Oxr1 and Rtc5. Cells expressing C-terminally mNeonGreen tagged Stv1 (Stv1-mNG) in a control strain and in OXR1 and RTC5 deletion strains. Sec7 was C-terminally tagged with 2xmKate2 (Sec7-2xmK2) as a late-Golgi marker and Pfa3 was tagged with the HaloTag (Pfa3-HT) and stained with JFX650 as a vacuole membrane marker. Panel A shows representative images, the scale bar represents 5 µm. Panels B and C show a co-localization analysis of Stv1-mNG with Pfa3-HT (B) or Sec7-2xmK2 (C) using Mandeŕs coefficients M1 and M2 for the overlap of the two signals. Each circle represents a single cell, the squares represent the average of each of three independent experiments, and the diamonds the overall average with error bars representing standard deviation. The statistical comparison was performed by a one-way ANOVA among the means for each experiment, followed by a Tukey post-hoc test. The P-values shown represent the comparison between control and oxr1 Δ cells, the difference between wt and rtc5 Δ cells was non-significant in all cases. (D and E) Analysis of the intensity of Stv1-mNG signal in late-Golgi compartments (D) or in whole cells (E). Analysis of the intensity of Stv1-mNG in the same experiment shown in panel A. The mean intensity of the Stv1-mNG signal was measured in regions of interest (ROIs) representing the whole cell in an equatorial plane or in 3D ROIs representing late-Golgi compartments defined as structures positive for Sec7-2xmK2 signals. Each colored circle represents a single cell, and each black circle represents the mean of each of three independent experiments. Statistical comparisons were performed by a one-way ANOVA among the means for each experiment, followed by a Tukey post-hoc test. (F and G) Stv1(1-452)-mNeonGreen partially re-localizes to the vacuole in the absence of Oxr1. Fluorescence microscopy analysis of Stv1(1-452)-mNG localization in a control strain and in a strain lacking OXR1 , together with Pfa3-HT as a vacuole membrane marker. Panel F shows representative images, with a scale bar representing 5 µm. Panel G shows co-localization analysis using Mandeŕs M1 and M2 coefficients as described for panels B and C. (H) Diagram summarizing the in vivo roles yeast TLDc domain-containing proteins Oxr1 and Rtc5 with respect to the V-ATPase. Rtc5 localizes to the vacuole membrane based on its N-terminal myristoylation and interaction with the assembled V-ATPase complex. Both proteins favor disassembly of the complex, counteracting the role of the RAVE complex. Finally, Oxr1 is necessary for the retention of Stv1-containing V-ATPases in the late Golgi or endosomal compartments.

Article Snippet: Rtc5 is a protein of unknown function, predicted to contain a TLDc domain ( T re2/Bub2/Cdc16, L ysM, d omain c atalytic, Pfam PF07534, InterPro IPR006571, Prosite PS51886, SMART SM00584).

Techniques: Fluorescence, Microscopy, Expressing, Control, Marker, Staining, Membrane, Standard Deviation, Comparison, In Vivo