Sequestration from Protease Adaptor Confers Differential Stability to Protease Substrate  Jinki Yeom, Kyle J. Wayne, Eduardo A. Groisman  Molecular Cell  Volume 66, Issue 2, Pages 234-246.e5 (April 2017) DOI: 10.1016/j.molcel.2017.03.009 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Differential Stability of Protease Substrate by Sequestration from Protease Adaptor (A–C) Substrate specificity modulation mechanism via competition between an adaptor protein and a specific adaptor competitor. The ClpS adaptor recognizes the substrates Oat and PhoP and delivers them to the ClpAP protease, which degrades them. The MgtC protein competes with ClpS for PhoP, thereby protecting PhoP, but not Oat, from ClpS-dependent ClpAP-mediated proteolysis. The western blot and SDS-PAGE analysis for the time course in vitro degradation of PhoP (B) or heat aggregated Mdh (C) are shown. PhoP (0.2 μM) and Mdh (0.5 μM) were mixed with ClpAP in the absence or presence of ClpS (0.5 μM) and/or 3 μL in vitro of synthesized MgtC. Reactions contained 2 μL in vitro of synthesized ClpP and ClpA. All of the reactions were carried out at 30°C for the indicated times in the presence of an ATP regeneration system and started by the addition of substrates. After incubation, protein amounts were determined by anti-PhoP, anti-MgtC, and anti-Mdh antibodies and Coomassie-stained band following separation by 4%–12% SDS-PAGE gel. The data are representative of three independent experiments, which gave similar results. See also Figures S1 and S3. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The PhoP-Activated MgtC Protein Protects PhoP from ClpS-Mediated Proteolysis (A) Western blot analysis of crude extracts prepared from wild-type (14028s), clpA (JY650), clpS (JY651), and clpX (JY649) Salmonella grown under low Mg2+ conditions. The samples were analyzed using anti-PhoP and anti-AtpB antibodies. The data are representative of three independent experiments, which gave similar results. (B) Western blot analysis of crude extracts prepared from wild-type (14028s), clpS (JY651) and clpS Salmonella with no plasmid, with the plasmid vector (vector, pUHE-21-2-lacIq), or with a clpS-expressing plasmid (pclpS; pUHE-clpS). clpS transcription from pClpS was induced with IPTG (100 μM). The samples were analyzed using anti-PhoP and anti-OmpA antibodies. The samples were loaded on 4%–12% NuPAGE gels with normalization via cell abundance with optical density. The data are representative of three independent experiments, which gave similar results. (C) The PhoP half-life was determined in wild-type (14028s), clpA (JY650), clpS (JY651), and clpX (JY649) Salmonella. Protein synthesis was inhibited with chloramphenicol (1 mg/mL). The samples were removed at the indicated time points and analyzed by western blotting with anti-PhoP antibodies. The PhoP half-lives (t1/2) were calculated by regression analysis of the exponential decay of PhoP. There were 5-fold less protein that was loaded for the samples from the clpS and clpA strains than for the samples from the wild-type and clpX strains. (D) Western blot analysis of crude extracts prepared from wild-type (14028s), mgtC (EL4), clpS (JY651), clpS mgtC (JY655), clpX (JY649), clpX mgtC (JY652), clpA (JY650), and clpA mgtC (JY653) Salmonella. The samples were analyzed using anti-PhoP and anti-AtpB antibodies. The data are representative of three independent experiments, which gave similar results. (E) PhoP stability was determined in wild-type (14028s) and mgtC (EL4) Salmonella. (F) PhoP stability was determined in clpS (JY651) and clpS mgtC (JY655) Salmonella. The samples were analyzed using anti-PhoP and anti-AtpB antibodies. Five-fold more protein was loaded for the mgtC samples than for the wild-type and mgtC/clpS samples (E and F). The bacteria were grown in N-minimal media (pH 7.7) containing 15 μM MgCl2 for 6 hr. See also Figures S1 and S2. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The PhoP and ClpS Proteins Interact In Vitro and In Vivo (A) Interaction between the MgtC-FLAG, ClpS-FLAG, and AtpB-FLAG proteins synthesized using an in vitro transcription/translation system. The proteins were incubated with purified PhoP-HA protein at room temperature for 2 hr and then incubated with anti-FLAG and anti-HA magnetic beads for an additional 2 hr. The immunoprecipitated samples were analyzed using anti-FLAG and anti-HA antibodies. The data are representative of two independent experiments, which gave similar results. The boxes highlight relevant bands. (B) A pull-down assay was performed in wild-type (14028s), phoP-HA (EG13918), clpS-FLAG (JY674), and phoP-HA/clpS-FLAG (JY676) Salmonella following growth in N-minimal media (pH 7.7) containing 15 μM MgCl2 for 6 hr. The lysed cells were incubated with anti-FLAG and anti-HA beads overnight. The samples were loaded onto 4%–12% NuPAGE gels and analyzed using anti-HA and anti-FLAG antibodies. The data are representative of two independent experiments, which gave similar results. The boxes highlight relevant bands. See also Figure S2. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 The Five N-Terminal Amino Acids of PhoP Are Required for Protection by MgtC (A) In vitro protein-protein interactions examined by co-immunoprecipitation using MgtC-FLAG, PhoQ-FLAG, YqjA-FLAG, PhoP-HA, and PhoP-HA derivative lacking amino acids 2 to 4 (4a). All of the proteins were synthesized using an in vitro transcription/translation system incubated at room temperature for 2 hr and then incubated with anti-FLAG or anti-HA magnetic beads for an additional 2 hr. The immunoprecipitated samples with FLAG and HA magnetic beads were analyzed by western blotting using anti-HA or anti-FLAG antibodies. The boxes highlight relevant bands. (B) Fluorescence of wild-type (14028s) and mgtC (EL4) Salmonella harboring plasmids expressing PhoP-GFP chimeric proteins (pUHE-phoP-11aa-gfp, pUHE-phoP-9aa-gfp, pUHE-phoP-7aa-gfp, pUHE-phoP-5aa-gfp, and pUHE-phoP-3aa-gfp). A schematic of chimeric proteins with GFP indicated in green and PhoP in blue is shown (top). The fluorescence normalized by optical density is shown (bottom). Shown are the mean and SD from four independent experiments, which gave similar results. (C) Pull-down assay of wild-type (14028s), phoP-five amino acids-GFP (KW64), mgtC-FLAG (EG16539), and phoP-five amino acids-GFP mgtC-FLAG (JY610) Salmonella. The samples were analyzed with anti-GFP and anti-FLAG antibodies. The boxes highlight relevant bands. The data are representative of two independent experiments, which gave similar results. (D) Western blot analysis of wild-type (14028s) and mgtC (EL4) Salmonella carrying the CAT-expressing plasmids (pUHE-cat, pUHE-phoP-5aa-cat, and pUHE-cat-phoP-5aa) using anti-CAT and anti-GroEL antibodies. A schematic of CAT and chimeric proteins with CAT indicated in red and PhoP in blue is shown (top). The data are representative of three independent experiments, which gave similar results. (E) Pull-down assay using wild-type (14028s) and clpS-FLAG (JY674) Salmonella, and derivatives of the latter strain harboring the GFP-expressing plasmids pUHE-phoP-5aa-gfp or pUHE-phoP-3aa-gfp. The samples were analyzed using anti-GFP and anti-FLAG antibodies. The boxes highlight relevant bands. The data are representative of two independent experiments, which gave similar results. The bacteria were grown in N-minimal media (pH 7.7) containing 15 μM MgCl2 and 0.1 mM IPTG for plasmids for 6 hr. All of the pull-down assay samples were incubated with magnetic beads at 4°C overnight. See also Figure S4. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 MgtC Outcompetes ClpS for Interaction with PhoP (A and B) In vitro competition by a pull-down assay using PhoP-HA, ClpS-FLAG, and MgtC-FLAG proteins. (A) Interaction between PhoP-HA and ClpS-FLAG proteins under increasing MgtC-FLAG amounts. (B) Interaction between PhoP-HA and MgtC-FLAG proteins under increasing ClpS-FLAG amounts. (C) Dissociation curves of MgtC-FLAG (red line) and ClpS-FLAG (blue line) from PhoP-HA (from A and B; see STAR Methods). The IC50 corresponds to the concentration at which half of the proteins are dissociated from PhoP-HA. Shown are the mean and SD from three independent experiments. (D) In vivo competition for binding to PhoP-HA in wild-type (14028s), phoP-HA clpA::cm (JY590), mgtC-FLAG (EG16539), phoP-HA clpA::cm mgtC-FLAG (JY591), and phoP-HA clpA::cm mgtC-FLAG Salmonella harboring the clpS-expressing plasmid (pUHE-clpS). The bacteria were grown in N-minimal media (pH 7.7) containing 15 μM MgCl2 and IPTG (0.05, 0.2, and 0.5 mM) for 6 hr. The data are representative of two independent experiments, which gave similar results. (E) Levels of the MgtC, ClpS, and PhoP proteins determined by western blot analysis of crude extracts from phoP-HA/clpS-FLAG (JY676) Salmonella following growth under non-inducing (10 mM MgCl2, time zero) and inducing (15 μM MgCl2) conditions for 2.5, 3.5, 4.5, 5.5, 6.5, 8.5, or 24 hr. The numbers of protein molecules were calculated using purified proteins (see STAR Methods). Shown are the mean and SD from three independent experiments. See also Figures S5 and S6. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 MgtC Increases the Levels of Active PhoP Protein (A) Half-life of PhoP-HA in phoP-HA (EG13918) Salmonella grown in N-minimal media with 15 μM MgCl2 for 2 and 5 hr. The protein synthesis was inhibited by chloramphenicol (1 mg/mL). The samples were removed at the indicated times and analyzed by western blotting with anti-HA antibodies. There were 3-fold more protein that was loaded in the 2 hr samples than in the 5 hr samples. The half-lives (t1/2) were calculated by regression analysis of the exponential decay of PhoP. (B) Fold change in the mRNA levels of the PhoP-activated pagC and pgtE genes produced by wild-type (14028s) and mgtC (EL4) Salmonella. The fold change was calculated by dividing the mRNA levels produced by wild-type Salmonella by those produced by the mgtC mutant. The bacteria were grown in N-minimal media (pH 7.7) containing 10 μM MgCl2 for 2 and 5 hr. (C) Fold change in the mRNA levels of 21 PhoP-activated genes produced by wild-type (14028s) and mgtC (EL4) Salmonella. The fold change was calculated by dividing the mRNA levels produced by wild-type Salmonella by those produced by the mgtC mutant. mRNA levels of target genes were normalized to those of the ompA gene (15054/15055). (D and E) mRNA levels of the pagC (D) and pagK (E) genes produced by wild-type (14028s), mgtC (EL4), clpS (JY651), clpS mgtC (JY655), clpX (JY649), and clpX mgtC (JY652) Salmonella following growth in N-minimal medium (pH 7.7), with 10 μM MgCl2 at 37°C for 5 hr. mRNA levels were normalized to those of the ompA gene. The primers used in qRT-PCR are presented in Table S3. For all qRT-PCR analysis, shown are the mean and SD from three independent experiments. See also Figure S7. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 Removal or Substitution of N-Terminal Residues Renders PhoP Stability Independent of MgtC and ClpS, and MgtC Does Not Regulate Proteolysis of the ClpS Client Oat (A) Protein levels of PhoP-HA in wild-type (14028s), mgtC (EL4), clpS (JY651), mgtC clpS (JY655), phoP (MS7953s), and phoP mgtC (EG16743) Salmonella harboring a plasmid expressing phoP-HA (pphoP-HA; pUHE-phoP-HA) or a derivative lacking amino acids 2 to 5 (pphoP-4aa-HA; pUHE-phoP-5aa-HA). The bacteria were grown in N-minimal media (pH 7.7) containing 15 μM MgCl2 and 100 μM IPTG for 6 hr. The samples were loaded on 4%–12% NuPAGE gels with normalization by cell abundance with optical density and analyzed using anti-HA and anti-OmpA antibodies. The data are representative of two independent experiments, which gave similar results. (B) Protein levels of PhoP-HA in wild-type (14028s), mgtC (EL4), and clpS (JY651) Salmonella harboring a plasmid expressing HA-tagged PhoP (pphoP-HA; pUHE-phoP-HA) or a variant with the Leu4Ala substitution (pphoP-L4A-HA; pUHE-phoP-L4A-HA). The bacteria were grown as indicated in (A) with 300 μM IPTG. The samples were loaded on 4%–12% NuPAGE gels with normalization by cell abundance with optical density and analyzed using anti-HA and anti-OmpA antibodies. The data are representative of two independent experiments, which gave similar results. (C) Protein levels of the Oat protein, in oat-FLAG (JY655), oat-FLAG clpS (JY657), and oat-FLAG clpS mgtC (JY697) Salmonella harboring no plasmid, the plasmid vector (pUHE-21-2-lacIq), or a clpS-expressing plasmid (pclpS; pUHE-clpS). The bacteria were grown as indicated in (A). The samples were loaded on 4%–12% NuPAGE gels with normalization by cell abundance and analyzed using anti-FLAG, anti-PhoP, and anti-OmpA antibodies. The data are representative of two independent experiments, which gave similar results. See also Figure S1. Molecular Cell 2017 66, 234-246.e5DOI: (10.1016/j.molcel.2017.03.009) Copyright © 2017 Elsevier Inc. Terms and Conditions