Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms
- ETH Zurich, Switzerland
- Paul Scherrer Institute, ETH Zurich, Switzerland
Figures
Figure 1 with 1 supplement
Bioinformatical analysis of actinobacterial Cpa.
(A) Phylogenetic relationships of different members of the Cdc48 protein family to the novel actinobacterial homolog. The peroxisome biogenesis factor 1 and 6 families (PEX1 and PEX6) are depicted in magenta, spermatogenesis-associated factor (Spaf) and Spaf-like families are colored grey, the archaeal Cdc48 homolog family is shown in green and N-ethylmaleimide-sensitive fusion protein (NSF) family in red. The Cdc48-like protein of actinobacteria (Cpa) forms a separate tight cluster (blue), while sporadically occurring homologs in other bacteria fall within the archaeal cluster (grey branches within the green cluster). (B) Occurrence and arrangement of the cpa gene locus in selected actinobacterial genomes. (C) Domain arrangment of Cdc48-like protein. The protein features an N-terminal domain (N-domain, grey) followed by two consecutive AAA modules (D1 and D2, blue). Each of the modules can be further subdivided into a P-loop NTPase domain and an α-helical subdomain. Conserved motifs are abbreviated as: A – Walker A motif, B – Walker B motif, SN – sensor asparagine, R – arginine finger. Organism names are abbreviated as follows: Mtub – Mycobacterium tuberculosis, Rery – Rhodococcus erythropolis, Sery – Saccharopolyspora erythraea, Stro – Salinispora tropica, Mcar – Micromonospora carbonacea, Scoe – Streptomyces coelicolor, Tfus – Thermobifida fusca, Mlut – Micrococcus luteus, Bado – Bifidobacterium adolescentis, Krhi – Kocuria rhizophila.
Figure 1—figure supplement 1
Principal component analysis of Cdc48 family protein sequences.
(A) 80% of the total variance between sequences can be explained by the first 10 principal components (PCs). The fraction of variance explained by distinct PCs is shown as a bar diagram (in blue) and the cumulative variance is shown as a red line. (B) PCA scores plot of the first two components. Cdc48 family proteins group into discrete clusters in accordance with their previously assigned category. The first principal component allows for the best distinction between NSF and Spaf types of sequences from the rest of the family members, while the second PC can be used to best distinguish between eukaryotic p97, archaeal/bacterial Cdc48(-like) sequences, PEX1 and PEX6 as well as Spaf-like proteins. (C) PCA loadings plot of the first two principal components. The dots colored in red represent the alignment positions for which the distance to 0 on the second PC loading exceeded the cutoff of 30% of the maximum distance (positions with most variance) while the dots colored in blue represent positions where the cutoff was set to 10% and lower (positions with least variance). (D) Structural model of the rhodococcal Cpa generated using human p97 as a template. Positions with most variance (as shown in panel C) are colored red and positions with least variance (most conserved) are colored in blue. The Walker A and B motifs, as well as arginine finger and sensor asparagine are shown using stick representation and colored green when considered conserved and red when considered variable. Top: protomer view with locations of the N-domain, D1- and D2-modules indicated. Bottom: hexamer view (the front protomer not shown). R – arginine finger, sN – sensor asparagine, A – alanine. The designation X→Y denotes substitution of amino acid X to amino acid Y in the original sequence.
Figure 2 with 2 supplements
Cpa forms hexamers in presence of nucleotide.
(A) Gel filtration profiles of 30 µM rhodococcal Cpa in absence of nucleotide (black line) and in presence of 2 mM ADP-AlFx (red line). The peak at ~16 ml corresponds to a monomer while the peak at ~13.5 ml corresponds to a hexameric assembly. (B) SEC-MALS profile of rhodococcal Cpa in presence of 2 mM ADP-AlFx. The red line represents refractive index, the black line corresponds to the light scattering signal and dark-blue triangles represent the fitting of the molar mass. (C) A representative negative-staining electron micrograph of a double Walker B mutant of Cpa shows ring-shaped particles in the presence of ATP. Upper right panel shows a 2D class average of Cpa particles with a hexameric ring structure. Scale bar represents 150 Å.
Figure 2—figure supplement 1
Electron microscopy image averaging.
2D class averages of Cpa particles show predominantly ring-shaped particles. Scale bar represents 150 Å.
Figure 2—figure supplement 2
Mycobacterial Cpa forms hexamers in the presence of ADP-AlFx and readily hydrolyses ATP.
(A) Gel filtration profiles of 24 µM MsmCpa in absence of nucleotide (black line) and in presence of 2 mM ADP-AlFx (red line). Similarly to the rhodococcal protein, the peak at ~16 ml corresponds to a monomer/dimer mixture while the peak at ~13.5 ml corresponds to a hexameric assembly. (B) ATPase activity of mycobacterial Cpa is five times higher at 37°C compared to 28°C. At 37°C the enzyme hydrolyzed ATP with a turnover number of 42.5 ± 3.2 min−1 hexamer−1 while at 28°C only 7.9 ± 2.9 min−1 hexamer−1 are turned over. Mean ± SEM of three replicates are shown.
Figure 3
Cpa complex assembly and ATPase activity are strongly influenced by pH and ionic strength.
(A) ATPase activity is enhanced with increasing pH and decreasing KCl concentration. The activity was measured at 28°C using 1.5 µM RerCpa (protomer). For readability reasons, the errors of the measurements are not shown – in all cases the error did not exceed 5% of the measured value. (B) Increasing ionic strength slows down Cpa complex formation. Assembly reactions were prepared using 9 µM RerCpa in a buffer containing increasing KCl concentrations (0–400 mM), incubated at 18°C and injected onto a SEC column at the indicated time points.
Figure 4 with 1 supplement
Cpa forms a complex with the 20S proteasomal core particle.
(A) Principle of the bacterial adenylate cyclase two-hybrid system. The cpa gene was fused to the T25 subdomain of adenylate cyclase while the proteasome was fused to the adenylate cyclase T18 subdomain. Interaction of the two complexes in E. coli ΔcyaA gives rise to increased β-galactosidase activity. (B) Both MtbCpa wt as well as MtbCpa lacking the five C-terminal residues when coexpressed with CP produce an increase of β-galactosidase activity as compared to the negative control, suggesting Cpa-CP complex formation (statistical significance was tested using two-way ANOVA). (C) Electron micrograph of negatively stained Cpa particles and 20S proteasomes. White arrows indicate side views of stacked complexes between Cpa and the 20S proteasome, blue arrows point at free Cpa rings and yellow arrows indicate uncapped 20S CP side views.
Figure 4—figure supplement 1
Representative electron micrograph of negatively stained Cpa particles and 20S proteasomes.
White arrows indicate side views of stacked complexes between Cpa and the 20S proteasome.
Figure 5
Cpa competes with Mpa for the binding site on the proteasomal core particle.
(A) Rhodococcal Cpa slows down the degradation of PanB-Pup by mycobacterial Mpa-20S CP complex (concentrations: 4 µM MtbPanB-Pup (protomer), 12 µM RerCpa (protomer), 0.2 µM Mpa (hexamer), 0.1 µM Mtb∆7PrcAB complex). (B) Rhodococcal Cpa slows down the degradation of linear Pup-GFP fusion by rhodococcal ARC-20S CP complex in a concentration-dependent manner (concentrations: 0.5 µM RerPup-GFP, 0.6/1.5/3.0 µM RerCpa (protomer), 50 nM RerArc (hexamer), 25 nM Rer∆7PrcAB complex).
Figure 6
Microscale thermophoresis measurement of dissociation constant between RerCpa and Mtb20S CP.
(A) Binding curves of RerCpa vs. closed- (red) and open-gate (black) MtbCP in presence of ATP. The gray triangles represent a dataset recorded for Cpa and open-gate proteasome in absence of nucleotide. (B) Dissociation constants and Hill coefficients for the two measured datasets.
Figure 7 with 1 supplement
M. smegmatis Δcpa shows normal growth behavior under standard culture conditions but displays a slight growth defect during carbon starvation.
(A) Msm parent and knockout cells were cultured in Middlebrook 7H9 medium at 37°C and cell density was measured at 600 nm at the indicated time points. Both strains showed identical growth behavior indicating that Cpa is dispensable during standard cell culture conditions. (B) cpa-knockout cells show impaired growth in the same medium devoid of glycerol as a main carbon source. Both strains were cultured in a minimal medium in absence of glycerol at 37°C. Both growth curves are representative of three or more independent experiments with the mean values ± SD plotted.
Figure 7—figure supplement 1
Western blot analysis of the Msm ∆cpa strain.
An anti-MsmCpa antibody (bottom) was used to show the presence (left lane, wild-type M. smegmatis) or the absence (Msm ∆cpa) of the Cpa protein in the two Msm strains used in this study. An antibody against the E. coli RNA polymerase subunit β was used as a loading control (top).
Figure 8 with 1 supplement
The cpa gene is co-transcribed together with neighboring psd and pssA genes.
(A) Organization of the cpa gene locus in Msm and design of probes used to test for gene co-transcription. (B) Visualization of all six probes amplified from cDNA produced from total RNA using a cpa-specific primer (middle of the gel). A set of reactions without reverse transcriptase was included to test for presence of contaminating genomic DNA (NTC – no template control; left part of the gel) as well as standard PCR with genomic DNA as a template to visualize the expected length of all probes (right part of the gel).
Figure 8—figure supplement 1
M. smegmatis cells lacking cpa do not exhibit significant differences in size or shape to wild-type cells either under standard conditions or when grown in absence of glycerol.
(A) Light microscopy images of M. smegmatis wild-type and ∆cpa cells during growth in presence (top) and absence (bottom) of glycerol as a main carbon source. (B) Statistical analysis of the average cell length during carbon starvation. A histogram depicting the distribution of cell length (in pixels) is shown on the left and the average cell length for both strains is shown on the right. The error bars represent standard error of the mean (SEM). The cell length difference is statistically significant at the p-value of 0.0001 (unpaired two-tailed t-test).
Figure 9 with 2 supplements
Label-free quantification mass spectrometry comparison of M. smegmatis WT with Δcpa proteome.
(A) Results of protein abundance comparison between the wt and the cpa-knockout strain using LFQ-MS (see Figure 9—source data 1). To filter the statistically-significant proteins, the p-value threshold was set to 0.05 (horizontal dashed line) and the fold-change threshold to 1.5 (vertical dashed lines). Under standard growth conditions (left plot), 49 proteins were found to accumulate (right side of the plot) and 45 proteins were found to be decreased (left side of the plot) in the knockout cells. Under carbon starvation (right plot), 254 proteins accumulated and 251 proteins were depleted in the knockout cells. (B) Those identified proteins for which a functional association was known or predicted, classification into functional classes using the COG classification system was carried out. Class abbreviations are as follows: C – energy production and conversion, D – cell cycle control and cell division, E – amino acid metabolism and transport, F – nucleotide metabolism and transport, G – carbohydrate metabolism and transport, H – coenzyme metabolism, I – lipid metabolism, J – translation/ribosomal structure and biogenesis, K – transcription, L – replication/recombination/repair, M – cell wall/membrane/envelope biogenesis, O – post-translational modification/protein turnover/chaperone functions, P – inorganic ion transport and metabolism, Q – secondary metabolites biosynthesis and catabolism, T – signal transduction, V – defense mechanisms. Proteins without an assigned class or assigned to class S (function unknown) were not included in the plot.
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Figure 9—source data 1
Source data file for Figure 9.
- https://doi.org/10.7554/eLife.34055.020
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Figure 9—source data 2
Source data file for Figure 9.
- https://doi.org/10.7554/eLife.34055.021
Figure 9—figure supplement 1
STRING interaction network of the proteins that accumulated in the Cpa-knockout strain during carbon starvation.
The blue circles represent ribosomal proteins and the red circles correspond to membrane proteins. The entire network represents proteins identified by LFQ-MS as having increased levels in the cpa-knockout strain under carbon limitation. Edge thickness of lines between the protein nodes corresponds to interaction confidence, according to STRING confidence scores.
Figure 9—figure supplement 2
Ribosomal proteins accumulating in the cpa-knockout cells during starvation mapped onto the structure of the 70S mycobacterial ribosome.
(A) Proteins colored green belong to the 50S subunit of the ribosome, while the blue ones belong to the 30S subunit. Red color indicates proteins that accumulated in the cpa-knockout strain during carbon starvation. For clarity, rRNA is shown in semi-transparent grey cartoon representation. The figure was generated using the recent structure of the 70S ribosome from M. smegmatis (PDB: 5O61, (Hentschel et al., 2017)). (B) Depiction of accumulating ribosomal proteins (red) in the context of the KEGG pathway map for the assembled ribosome (Kanehisa et al., 2016). Boxes framed in bold represent proteins identified in at least two out of three replicates of the co-immunoprecipitation experiment.
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene | mKO2 | Thermo Fisher Scientific | NCBI accession: AB359188 | GeneArt synthetic construct |
| Strain (Escherichia coli) | E. coli Tuner(DE3) | EMD-Millipore | ||
| Strain (Mycobacterium smegmatis mc(2) 155) | Wild-type strain (Msm WT) | ATCC | ATCC: 700084 | |
| Genetic reagent (Mycobacterium smegmatis mc(2) 155) | Msm ∆cpa | This paper | Unmarked cpa deletion strain | |
| Antibody | anti-RpoBEc | BioLegend | BioLegend clone: 8RB13 | Antibody against beta-subunit of RNA polymerase from E. coli; used at dilution 1:1000 |
| Antibody | anti-CpaMsm | This paper | Rabbit polyclonal antibody against full-length Cpa from M. smegmatis mc(2) 155; produced by BioGenes GmbH; used at dilution 1:40000 | |
| Recombinant DNA reagent | pET28a-His6- TEV-RerCpa | This paper | Plasmid for expression of rhodococcal Cpa | |
| Recombinant DNA reagent | pET28a-His6- TEV-MsmCpa | This paper | Plasmid for expression of mycobacterial Cpa | |
| Recombinant DNA reagent | pET28a-His6-TEV-Rer Cpa_D312N_E566Q | This paper | Plasmid for expression of double Walker B mutant of rhodococcal Cpa | |
| Recombinant DNA reagent | pET28a-His6-TEV -mKO2-RerCpa | This paper | Plasmid for expression of mKO2-Cpa fusion protein used in MST experiments | |
| Recombinant DNA reagent | pETDuet-RerPrcA -PrcB∆pro | This paper | Plasmid for expression of rhodococcal proteasome (closed-gate variant) | |
| Recombinant DNA reagent | pETDuet-RerPrcA∆7 -PrcB∆pro | This paper | Plasmid for expression of rhodococcal proteasome (open-gate variant) | |
| Recombinant DNA reagent | pET28a-His6-TEV-RerArc | This paper | Plasmid for expression of rhodococcal Arc | |
| Recombinant DNA reagent | pETDuet-MtbPanB-Strep | PMID: 20203624 | ||
| Recombinant DNA reagent | pTrc99-MtbMpa | PMID: 20203624 | ||
| Recombinant DNA reagent | pET20-His6-Trx- TEV-MtbPup-GFPuv | PMID: 20203624 | ||
| Recombinant DNA reagent | pKT25 | Euromedex | ||
| Recombinant DNA reagent | pUT18 | Euromedex | ||
| Recombinant DNA reagent | pKT25-zip | Euromedex | ||
| Recombinant DNA reagent | pUT18C-zip | Euromedex | ||
| Recombinant DNA reagent | pKT25-MtbCpa | This paper | Bacterial two-hybrid plasmid: T25-Cpa fusion | |
| Recombinant DNA reagent | pKT25-MtbCpa∆C5 | This paper | Bacterial two-hybrid plasmid: T25-Cpa fusion where Cpa is C-terminally truncated by five residues | |
| Recombinant DNA reagent | pUT18-MtbPrcA- PrcB∆pro | This paper | Bacterial two-hybrid plasmid: mycobacterial proteasome with the T18 subdomain fused to the C-terminus of PrcA | |
| Commercial assay or kit | Monolith NT.115 Premium Coated Capillaries | NanoTemper Technologies | MO-K005 | |
| Chemical compound, drug | 4-Methylumbelliferone | Sigma-Aldrich | M1381 | |
| Chemical compound, drug | 4-Methylumbelliferyl β-D-galactopyranoside | Sigma-Aldrich | M1633 | |
| Software, algorithm | STRING | PMID: 27924014 | ||
| Software, algorithm | DAVID | PMID: 22543366 | ||
| Software, algorithm | FastTree | PMID: 20224823 | ||
| Software, algorithm | SWISS-MODEL | PMID: 28874689 | ||
| Software, algorithm | PyMOL | Schrödinger, LLC | http://www.pymol.org |
Additional files
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Supplementary file 1
Supplementary tables.
Supplementary Table 1. List of proteins used for ClustalO alignment of the Cdc48 family (NCBI accession numbers). Supplementary Table 2. List of proteins accumulating during growth in the presence of glycerol in M. smegmatis Δcpa as compared to its parent strain by label-free quantification mass spectrometry. Supplementary Table 3. List of proteins depleted during growth in the presence of glycerol in M. smegmatis Δcpa as compared to its parent strain by label-free quantification mass spectrometry. Supplementary Table 4. List of proteins accumulating during growth in the absence of glycerol in M. smegmatis Δcpa as compared to its parent strain by label-free quantification mass spectrometry. Supplementary Table 5. List of proteins depleted during growth in the absence of glycerol in M. smegmatis Δcpa as compared to its parent strain by label-free quantification mass spectrometry.
- https://doi.org/10.7554/eLife.34055.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.34055.023
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Cdc48-like protein of actinobacteria (Cpa) is a novel proteasome interactor in mycobacteria and related organisms
eLife 7:e34055.
https://doi.org/10.7554/eLife.34055
