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

Journal: Cancers

Article Title: XPO1 E571K Mutation Modifies Exportin 1 Localisation and Interactome in B-Cell Lymphoma

doi: 10.3390/cancers12102829

Figure Lengend Snippet: Enrichment for XPO1 interactors identified in K1106 and MedB1 cells with STRING database.

Article Snippet: We classified XPO1-interacting proteins using the STRING database tools ( ) and found the same GO terms: biological process (BP), molecular function (MF) and cellular component (CC), and the same INTERPRO and SMART protein domains, confirming that both interactomes are similar in the two cell lines.

Techniques: Activity Assay, Membrane

Journal: Genetics

Article Title: OrthoList 2: A New Comparative Genomic Analysis of Human and Caenorhabditis elegans Genes

doi: 10.1534/genetics.118.301307

Figure Lengend Snippet: Workflow for genome analysis and generation of OL2. The workflow proceeded in four steps. Step 1: we addressed changes to gene models in the worm genome that have occurred since OL1 was published (File S1) to yield an updated OL1 (File S2). We also addressed changes to human gene predictions (File S3). Step 2: we queried updated versions of the orthology-prediction methods used in OL1 (see Table 1) to generate OL1.1 (File S4), and found that the number of worm genes added was within the parameters predicted by changes in individual programs (Table 2), whereas gene loss appeared to be buffered by combining results from the different methods (i.e., the meta-analysis approach). Step 3: we next added results from two additional orthology-prediction methods (see Table 1) and found that this had a low impact on the landscape of human–worm orthologs identified in OL1.1 (File S5). Finally, in step 4, we combined the genes identified by these two additional programs with OL1.1 to generate OL2 (File S5 and File S7). We note that genes that did not continue to be supported by orthology-prediction methods were retained as a legacy set present in the searchable database (File S6 and File S7). Both OL2 and the legacy set of genes were cross-referenced to the C. elegans feeding RNAi library, protein domain prediction databases (InterPro and SMART), and to a human disease association database (OMIM) to generate a final master list (File S7), which can be queried via the new Web-based tool found at http://ortholist.shaye-lab.org.

Article Snippet: Finally, we include links to SMART and InterPro protein domain descriptions, as well as to OMIM entries for human disease associations.

Techniques:

Journal: Genetics

Article Title: OrthoList 2: A New Comparative Genomic Analysis of Human and Caenorhabditis elegans Genes

doi: 10.1534/genetics.118.301307

Figure Lengend Snippet: OL2 query interface. (A) Input page at http://ortholist.shaye-lab.org. Users can select which fields to search (human and worm IDs, SMART or InterPro protein domains, and disease phenotypes described in OMIM); whether to set a threshold for orthology support (see main text); and whether partial matches should be allowed, which is useful when users want to find all members of a similarly named gene family (e.g., input “Notch” to find all human Notch family members). (B) Sample results page for the gene let-60, with a search conducted using the default settings, returning a set of Ras orthologs consistent with its sequence and genetic validation in a canonical Ras pathway (Han and Sternberg 1990; Sundaram 2013). The results page contains links for viewing additional information about results and for exporting results to a comma-separated value (CSV) spreadsheet.

Article Snippet: Finally, we include links to SMART and InterPro protein domain descriptions, as well as to OMIM entries for human disease associations.

Techniques: Sequencing, Biomarker Discovery