EMBO/soda-nel huggingface dataset:
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 1

Fig. 1: Quantitative proteomics reveals candidate interactors and substrates of rhomboid protease YqgP from B. subtilis.

(A) Schematic representation of the SILAC-based quantitative proteomics experiment in B. subtilis. Cells overexpressing active YqgP (BS50, Table EV2) or its catalytically dead mutant YqgP.S288A (BS51), both auxotrophic for lysine, were grown in parallel in "heavy" (containing 13C615N2- Lysine isotope) or "light" (containing stable 12C614N2- Lysine) M9 minimal medium, respectively. After mixing the cell cultures in the 1:1 ratio (based on OD600), cell suspension was lysed and the fraction enriched for transmembrane proteome was analysed in a GeLC-MS/MS experiment. Using bioinformatic analysis of the MS data, highest-scoring candidate substrates were further evaluated.

(B) Diagrammatic representation of a result of GeLC-MS/MS analysis of excised gel regions described in (A). Each coloured block depicts a quantified peptide at its amino acid position according to the horizontal axis. Possible ongoing proteolysis was identified by high abundance ratio of YqgP/YqgP.S288A for a given protein and lower apparent molecular weight than expected for the corresponding full-length protein, as estimated from the position of the respective gel slice relative to the molecular weight marker.

(C) Table of best substrate candidates of two GeLC experiments. In order to assess possible cleavage sites in combination with topology information, the QARIP software Ivankov et al., 2013() was used to summarize results from experiments 1 and 2. Abundance ratios (YqgP/YqgP.S288A) for the intracellular parts of proteins were calculated for each gel slice separately by QARIP from peptide ratios computed by MaxQuant Cox & Mann, 2008(). Substrate candidates highlighted in red were identified in both experiments.

(D) Schematic representation of the affinity co-purification experiment in B. subtilis to identify YqgP interactors.

(E) Results of MS analyses of affinity co-purification experiments in wild-type B. subtilis control (strain BTM2, Table EV2) and B. subtilis deficient in endogenous YqgP expressing the wild-type YqgP-sfGFP bait (strain BTM84, Table EV2) or the proteolytically inactive YqgP.S288A-sfGFP bait (strain BBM1, Table EV2). Proteins were considered as potential interactors of YqgP if they were identified only in both positive co-purifications with a minimum of 3 weighted spectral counts or enriched at least 5 times in positive bait samples compared to control ones based on weighted spectral counts. The protein highlighted in red was the only overlapping hit between the two proteomics approaches.
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 2

Fig. 2: YqgP cleaves the high-affinity magnesium transporter MgtE between its first and second transmembrane helices.

(A) Steady-state cleavage profile of endogenous MgtE processed by endogenous and ectopically overexpressed YqgP from the inducible Phyperspank promoter in living B. subtilis (BTM2 and BTM501, respectively, Table EV2), in minimal medium at low magnesium concentration (10 µM). Strain lacking YqgP (ΔyqgP, BTM78, Table EV2) was used as a control. Proteins were detected by immunoblotting with chemiluminescence detection.

(B) The cleavage is efficiently inhibited by 1 μM STS736, a specific peptidyl ketoamide rhomboid inhibitor Ticha et al., 2017b().

(C) To map the cleavage site region within endogenous MgtE (second lane from the left, from strain BTM501), MgtE-derived reference fragments encoding first 300, 315, 330 amino acids as well as full-length MgtE (black arrow), were in vitro transcribed and translated. Mobility of the N-terminal cleavage product (red arrow) of MgtE on SDS PAGE was compared to the mobilities of the translated reference fragments.

(D) Diagrammatic display of the mapping shows that YqgP cleaves MgtE in a periplasmic region between transmembrane helices 1 and 2 (red arrow).

Data information: In all panels, endogenous full-length MgtE is indicated by a black arrow, and N-terminal cleavage product by red arrows. Endogenous MgtE was visualised by anti-MgtE(2-275) (α-MgtE) and ectopic YqgP by anti-YqgP antibodies.
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 3

Fig. 3: Magnesium starvation and manganese excess activate cleavage of MgtE by YqgP, which is beneficial in manganese stress conditions.

(A) Detection and quantification of the cleavage of endogenous MgtE by YqgP in living B. subtilis cells (BS72, Table EV2) depending on the concentrations of magnesium and manganese ions. Cells were cultivated in glucose M9 minimal medium with limiting (0.01 mM) or high (1 mM) concentration of MgSO4, in the presence or absence of 100 μM MnCl2, and analysed by western blotting with near-infrared detection (upper panel). Black arrow denotes full-length MgtE, and red arrow denotes its N-terminal cleavage product formed by YqgP. The corresponding fluorescence signals were quantified by densitometry, and are displayed as relative specific activity, which is substrate conversion normalized to enzyme expression level (lower panel).

(B) Growth curves of wild-type (BTM843, Table EV2), yqgP deficient (BTM844, Table EV2) and rescue (BTM845, Table EV2) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese stress elicited by adding 75 µM MnSO4 in mid-exponential phase (stress-phase denoted by blueish background). All strains further contain a deletion in the putative manganese efflux pump MntP (ΔywlD, Table EV2). Bottom panel shows that manganese is more toxic in the yqgP deficient strain than in the wild type strain, and that reintroduction of YqgP rescues fitness during manganese stress to above-wild-type level. Top panel shows no difference between the strains in the absence of manganese stress. Data are shown as individual datapoints from three independent experiments overlaid with dashed line connecting average values from each time-point, which illustrates the reproducibility of the assay.

(C) Top panel: growth curves of wild-type (BTM843), yqgP deficient (BTM844) and rescue (BTM845) strains of B. subtilis in M9 minimal medium with limiting magnesium (0.01 mM MgSO4), exposed to manganese shock elicited by adding 75 µM MnSO4 in mid-exponential phase. Bottom panel: manganese toxicity is prevented by further adding 5 mM magnesium (MgSO4) in otherwise identical conditions.

(D) Inhibition of YqgP by a rhomboid-specific peptidyl ketoamide inhibitor (STS736, i.e. compound 9 from Ticha et al., 2017b()) abolishes the YqgP induced fitness of B. subtilis under manganese stress, in a dose-dependent manner, both with endogenous YqgP (top panel) and overexpressed YqgP (bottom panel). Media and growth conditions were identical to those used in panel (B).

(E) Overexpression of heterologous MgtE inhibits growth of yqgP deficient strain (BTM610, Table EV2) in rich LB medium supplemented with 75 μM MnSO4. Cell fitness is improved by overexpression of YqgP (BTM611, Table EV2) or its catalytically dead mutant YqgP.S288A (BTM612, Table EV2). For clarity, for panels C - E, representative experiments of 2-3 independent biological replicates are shown.
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 4

Fig. 4: The N-terminal extramembrane intracellular domain of YqgP mediates the manganese induced activation of MgtE cleavage.

(A) Schematic of YqgP domain architecture. Transmembrane helices are shown as grey boxes, and the position of the catalytic dyad of S288 and H339 within the transmembrane rhomboid core is displayed.

(B) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE and model chimeric substrate derived from Providencia stuartii TatA Stevenson et al., 2007Ticha et al., 2017aTicha et al., 2017b(, , ) (schemes in the middle) by ectopically expressed YqgP and its N- and C-terminally truncated variants (strains BS184-187, Table EV2) in living B. subtilis grown in rich LB medium. Black arrows indicate full-length substrates and red arrows indicate cleavage products.

(C) Similar analysis of the same strains but grown in minimal M9 medium (with 10 µM MgSO4) supplemented with increasing concentrations of MnCl­­2. Black arrows denote full-length substrate (MgtE), red arrows denote the cleavage products generated by YqgP.

(D) Western blots of four independent experiments shown in panel (C) were quantified by near-infrared fluorescence detection, quantified by densitometry and the results are displayed as fold activation of MgtE cleavage at 100 µM MnCl­­2 relative to 1 µM MnCl­­2 activity. Average values and all fours datapoints are plotted for each indicated YqgP variant.
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 5

Fig. 5: The N-terminal intracellular globular domain of YqgP binds divalent cations.

(A) Detection (left panel) and quantification (right panel) of steady-state conversion of endogenous MgtE by YqgP (strain BS72, Table EV2) grown in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (reference conditions) with the additions of 10 µM or 100 μM of a given divalent cation salt solution (MnCl2, ZnCl2, CoCl2, or NiCl2), or 50 µM and 500 μM for CaCl2. Western blots were quantified by near-infrared fluorescence detection (left), quantified by densitometry and displayed as relative specific activity (graph on the right), which is substrate conversion normalized to enzyme expression level. Black arrow denotes full-length substrate (MgtE), and red arrow denotes the N-terminal cleavage product(s) generated by YqgP. All bands originate from the same western blot and identical treatment series. Irrelevant lanes have been cropped out for clarity, and the source blot is available online as Source Data.

(B) Isothermal titration calorimetry of purified recombinant YqgPNTD and selected divalent cations.

(C) Cation toxicity assays and their relationship to YqgP activity. Wild type B. subtilis (BTM2, Table EV2), its variant lacking YqgP (ΔyqgP, BTM78, Table EV2), and the rescue strain ectopically expressing YqgP (BTM501, Table EV2) were cultivated in minimal M9 medium containing 10 μM MgSO4 and 1 μM each of MnCl2, ZnCl2, CoCl2, NiCl2 and CaCl2 (i.e. reference conditions), and in mid-exponential phase were stressed by the addition of either 70 µM MnCl2, 500 µM ZnCl2, 25 µM CoSO4 or 400 µM NiCl2 (pale blue area). YqgP activity specifically improved cell fitness during manganese and zinc stress, while it had no effect on growth of cells cultivated in the presence of high cobalt and nickel concentrations. Representative experiments of 2-3 independent replicates are shown.
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 6

Fig. 6: Solution NMR analysis reveals manganese-binding region in the N-terminal domain of YqgP.

(A) Solution NMR structure of cytosolic N-terminal domain of YqgP (YqgPNTD, amino acids 1-177), showing ensemble of 30 structures with lowest energy, and the structure with minimum energy displayed as cartoon.

(B) Electrostatic surface of YqgPNTD structure reveals highly negatively charged areas. Left view is oriented as the left view in panel (A).

(C) Chemical shift perturbation values and relative peak intensities for backbone resonances (15N and 1H) of YqgPNTD calculated for complexes of 400 μM YqgPNTD with 10 μM (light grey, light orange) or 40 μM MnCl2 (dark grey, dark orange bars). Only residues 20-100 of YqgP are displayed for simplicity. Residues identified as potential manganese-binding region based on their chemical shifts and intensity changes upon NMR titrations are depicted in orange. The X sign marks residues whose chemical shifts were not calculated due to the lack of resonance signal.

(D) Details of 2D 15N/1H HSQC spectra of free (green) and Mn-titrated YqgPNTD (10 μM MnCl2 in red and 40 μM MnCl2 in blue) for the residues marked in orange in panel (C). As a reference, residues marked in black do not display spectral changes upon titration, e.g. peak intensity does not change for S88N-H upon Mn titration (overlay of green, blue and red contours), but peak intensity is severely depleted for D29N-H upon Mn titration (absence of red contours). Full titration data are available in Dataset S3.

(E, F) Detection (E) and quantification (F) of steady-state conversions of endogenous MgtE by YqgP variants bearing single-point mutations in putative Mn-binding region (BS184; 187; 196-203, Table EV2), cultivated in modified M9 minimal medium supplemented by low (1 μM) or high (100 μM) MnCl2. Black arrow marks full-length MgtE, and red arrow marks its N-terminal cleavage product by YqgP.

(G) Overview of residues affected by Mn2+ binding to YqgPNTD mapped onto its solution NMR structure. The view is oriented as the left view in panels (A and B).
Rhomboid intramembrane protease YqgP licenses bacterial membrane protein quality control as adaptor of FtsH AAA protease (2019)
Figure 7

Fig. 7: The YqgP rhomboid functions as both a protease and a pseudoprotease adaptor of the AAA-protease FtsH to maintain proteostasis of MgtE.

(A) The proteolytically dead mutant YqgP.S288A behanes as a pseudoprotease and acts as a substrate adaptor of FtsH. Pattern of C-terminally truncated forms of endogenous MgtE formed in vivo in the presence of ectopically expressed active YqgP (strain BS72, Table EV2) or its inactive S288A mutant (BS73, Table EV2) in rich LB medium in the presence or absence of endogenous FtsH. Black arrow denotes the full-length MgtE substrate, and red arrows denote its cleavage products generated by YqgP. While the presence of the faster migrating YqgP-dependent products (full red arrows) do not depend on the presence of FtsH, the slower migrating product formed in the presence of YqgP.S288A (open red arrow) does, indicating that FtsH protease is responsible for the alternative cleavage of MgtE. The same arrow symbolics are valid throughout this figure.

(B) Mapping of the boundary of the FtsH generated proteolytic product of MgtE. Immunoblot comparing the SDS PAGE mobility of the N-terminal fragment of endogenous MgtE generated by FtsH in the presence of the pseudoprotease version of YqgP (catalytically dead YqgP.S288A, strains BTM78, BTM502, Table EV2) to the mobilities of in vitro translated reference fragments corresponding to MgtE 1-340, -355 and -370 shows that YqgP.S288A acts as an FtsH adaptor, and that FtsH processing of MgtE in the presence of YqgP.S288A stops at around amino acid 355, which is near or at the cytoplasmic end of TMH 3 of MgtE.

(C) Schematic depiction of the regions in MgtE defining its remaining cleavage products formed by FtsH and YqgP.

(D) In vivo cleavage of endogenous MgtE in the presence of ectopically expressed full-length YqgP.S288A (strain BS73), YqgPΔNTD.S288A (BS185, Table EV2) or YqgPΔCTD.S288A (BS186, Table EV2), in rich LB medium shows that YqgPNTD is necessary for the FtsH-dependent processing of MgtE.

(E) Analysis of the in vivo kinetics of formation and fate of MgtE cleavage products. B. subtilis strains expressing YqgP or its S288A mutant (strains BTM78; 501 and 502, Table EV2) in the presence or absence of endogenous FtsH protease (strains BTM795, BTM796 and BTM797, Table EV2) were cultured in LB medium and at early exponential phase, after having been expressing YqgP variants for 30 min, were either left grown untreated (as a control with ongoing translation) or treated with 20 μg/mL tetracycline to stop proteosynthesis (translation shut-off). At given time intervals afterwards, all cultures were analysed by α-MgtE western blotting with near infrared detection. Equal cell number (judged by OD600) was loaded into each lane. Black arrow marks full-length MgtE, red full arrows mark YqgP-dependent and red open arrows mark YqgP.S288A-dependent cleavage product of MgtE.
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 3

Figure 3. Histone humanization cause centromere dysfunction in yeast

(a) Centromeric nucleosome occupancy. The relative nucleosome occupancy plots are given for each strain in the top plot (grey, wildtype; red, DAD1E50D humanized; purple, scc4D65Y humanized). Values represent the chromosome mean occupancy 900 bp up and downstream of all 16 centromeres (each centromere occupancy values being an average of 3 biological replicates). Bottom plots, log2 ratio nucleosome occupancy enriched in humanized (green) and depleted in humanized (purple), each row represents one of the sixteen centromeric regions.

(b) Total RNA sequencing of cenRNAs in yeasts. Left; read counts per million tracks for CEN16 in wildtype (black, top) and DAD1E50D humanized (red, bottom) yeasts. Right; heatmap of cenRNAs transcripts per million counts across all chromosomes in wildtype and DAD1E50D humanized strains. Centromere VI is marked with an asterisk to indicate that the plasmid encoding the histone plasmid also encodes a CEN6 sequence, which reads likely emanant from (See Appendix Figure S3).

(c) RT-PCRs of the indicated RNAs. Lane 1 is genomic DNA, lane 2 is wildtype shuffle strain (yDT67), lane 3 is BY4742, lane 4 is DAD1E50D humanized, lane 5 is scc4D65Y humanized, lanes 6-9 are the same as 2-5, but without reverse transcriptase added, lane 10 is no DNA.

(d) Example images from strains with fluorescent tags on the microtubule organizing center (Spc110-GFP) and the kinetochore (Nuf2-RFP). Images were taken from cultures in mid-log phase of growth.

(e) Bar plots of the fraction of cells with clustered or declustered Nuf2-RFP foci. Brown shading indicates fraction of cells with correct clustering, and the purple shading indicates fraction of cells with declustered Nuf2-RFP foci. The number of cells analyzed for each genotype is shown below.
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 4

Figure 4. DASH/Dam1c mutations are sufficient for aneuploid reduction

(a) Genome-wide chromosome coverage maps for the parental (yeast histones) and the ancestral and evolved yHs13 strains (note the loss of aneuploid chromosome XII in the evolved isolate). Note that yHs13 is the lineage that evolved the mutation DAM1N80Y. Chromosome coverage copy was normalized as the log2 ratio of the average coverage of a 1 Kb window by the genome median coverage.

(b) Evolution of aneuploid chromosomes in histone-humanized strains. The number of aneuploid chromosomes is given for each of the indicated grouping of humanized strains. Sample size is given below each (number of histone-humanized lineages), Dunnett's tests of the mean differences between the three ancestral-evolved pairs are provided. Error bars report the standard deviation.

(c) Change in the average number of aneuploid chromosomes in humanized lineages with or without DASH/Dam1c mutations. Sample size is given below each, unpaired t-test of the mean difference is provided. Error bars report the standard deviation.

(d) Aneuploidy accumulation assay. Histone-humanization in the scc4D65Y genetic background results in persistent aneuploidy (red chromosomes), does the presence of a second mutation, DAD1E50D or DAM1N80Y, stop the accumulation of aneuploidies to maintain euploidy (grey chromosomes)?

(e) DASH mutants increase histone-humanization in the scc4D65Y background. Humanization rates of the scc4D65Y mutant with and without DAD1E50D or DAM1N80Y. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents one biological replicate (n = 8), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.

(f) Ploidy analysis by flow cytometry in the scc4D65Y humanized mutants from panel d. Flow cytometry data from humanized lineages yHs5 and yHs7 are shown for aneuploid and euploid controls, respectively.
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 5

Figure 5. DASH/Dam1c mutants rescue Aurora B kinase and SAC mutants

(a) Growth assays of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at permissive (24ºC) and non-permissive temperature (32ºC). ﻿Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto YPD solid media and grown at the indicated temperatures for four and three days, respectively.

(b) Flow cytometry analysis of wild type, ipl1-2 mutants, and ipl1-2 kinetochore double mutants at the permissive (24˚C) and non-permissive temperature (30ºC). Yeast cultures were grown to logarithmic phase at 24˚C and divided and grown at 24˚C and 30˚C for 6 hours before cells were collected and processed for DNA content analysis using Sytox Green stain.

(c) Growth assays of wild type, mad3∆ mutants, and mad3∆ DAD1E50D double mutants on YPD with and without 30 µg/ml benomyl. Ten-fold serial dilutions of ~1.0 OD600 yeast cultures were spotted onto the indicated solid media and grown at 30˚C for three days.

(d) Humanization of mad3∆ mutant and double mad3∆ DAD1E50D mutant. Each dot represents a biological replicate (n ≥ 3), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.
DASH/Dam1 complex mutants stabilize ploidy in histone-humanized yeast by weakening kinetochore-microtubule attachments (2022)
Figure 6

Figure 6. Molecular basis for suppression of histone humanization by DAD1E50D and DAM1N80Y

(a) Homology models of S. cerevisiae DASH/Dam1c complex. The decametric DASH/Dam1c Cyro-EM structure of C. thermophilum is shown, with Dad1 and Dam1 highlighted. The corresponding homology model is shown to the right, with various mutant models shown (with outlined WT residues for reference). Note the relative positioning of Dad1 residue 50 and Dam1 residue 80.

(b) Histone-humanization assay for various dam1N80X mutants. 5-FOA is used to counter-select the yeast histone plasmid, forcing growth with the human histone plasmid. Yeast were serially diluted from a starting culture of 1.0 OD600.

(c) Cartoon interactions for complementary pairings of Dad1 residue 50 and Dam1 residue 80.

(d) The dual mutant Dad1-Asp50 - Dam1-Gln80 DASH/Dam1c fails to humanize, while the single mutants readily humanize. Significance of the mean difference in 5-FOAR frequency was determined with the Mann-Whitney test. Each dot represents a biological replicate (n ≥ 4), central band represents the median, box extends from the 25th to 75th percentiles, and whiskers represent the minimum and maximum.

(e) Protein alignment of Dad1 orthologs, highlighting a conserved casein 1 kinase consensus phosphorylation site (yellow shaded region). Arrow indicates the mutant residue 50 of Dad1.