Site-2 Protease Substrate Specificity and Coupling in Trans by a PDZ

Site-2 protease substrate speciﬁcity and coupling in trans by a PDZ-substrate adapter protein

Jessica S. Schneidera,b, Shilpa P. Reddya, Hock Y. Ea, Henry W. Evansc, and Michael S. Glickmana,b,c,1

aImmunology Program, Sloan-Kettering Institute, New York, NY 10021; bProgram in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065; and cSummer Undergraduate Research Program, Gerstner Sloan-Kettering Graduate School of Biomedical Sciences, New York NY 10065

Edited by Michael S. Wolfe, Harvard Medical School and Brigham and Women’s Hospital, Boston, MA, and accepted by the Editorial Board October 14, 2013 (received for review April 1, 2013) Site-2 proteases (S2Ps) are intramembrane metalloproteases that Methanocaldococcus jannaschii S2P will cleave the Caeno- cleave transmembrane substrates in all domains of life. Many S2Ps, rhabditis elegans transmembrane protein Ced-9 (18). These results including human S2P and Mycobacterium tuberculosis Rip1, have strongly suggest that the substrate specificity of S2Ps is not de- multiple substrates in vivo, which are often transcriptional regu- termined by the primary sequence of the cleaved transmembrane lators. However, S2Ps will also cleave transmembrane sequences segment and that additional determinants of protease specificity of nonsubstrate proteins, suggesting additional specificity deter- exist in vivo (19). minants. Many S2Ps also contain a PDZ domain, the function of Another poorly understood feature of S2Ps is the mechanism which is poorly understood. Here, we identify an M. tuberculosis by which S1P and S2P cleavage events are coupled. Many S2Ps, protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges including E. coli RseP, Salmonella enterica RseP, Human S2P, between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), and M. tuberculosis Rip1, contain PDZ domains. Substantial a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. evidence indicates that these PDZ domains integrate S1 and site-2 In vivo analyses of Ppr1 function indicate that it prevents nonspe- cleavage by preventing the S2P from cleaving before the S1P. In cific activation of the Rip1 pathway while coupling Rip1 cleavage E. coli, RseP lacking its PDZ domain can complement the es- of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results sential function of RseP but decouples RseP activity from S1 support a model of S2P substrate specificity in which a substrate- –

proteolysis (20 23). However, the mechanism by which the PDZ MICROBIOLOGY specific adapter protein tethers the S2P to its substrate while hold- domain modulates S2P activity is not known. Despite the usual ing the protease inactive through its PDZ domain. function of PDZ domains in protein–protein interactions, and particularly in nucleating signaling complexes (24), no S2P PDZ intramembrane proteolysis | signal transduction | protein interactors have been identified. Tuberculosis | Sigma factor In M. tuberculosis, we have previously identified the Rip1 S2P as a major virulence determinant with three anti-sigma factor egulated intramembrane proteolysis (RIP) is a widely dis- substrates (3, 4). Here, we identify a Rip1-PDZ interacting pro- Rtributed mechanism of signal transduction across membranes tein, PDZ-interacting protease regulator (Ppr)1, which holds Rip1 in which specialized intramembrane cleaving proteases (iCLIPs) inactive while tethering Rip1 to one of its substrates, RsmA. After cleave the transmembrane segments of substrate proteins (1, 2). S1 cleavage, Ppr1 specifically facilitates Rip1 cleavage of RsmA The site-2 (S2) proteases (S2Ps) are one class of iCLIP that is but not other Rip1 substrates. We propose a model of S2P substrate widely distributed from bacteria to human cells and participate specificity and coupling in which the S2P PDZ domain nucleates in such diverse pathways as lipid biosynthesis (1, 3, 4), sporula- distinct signaling complexes through substrate-specific adapter tion (5), membrane stress (6, 7), alginate production (8), and polar proteins that both hold the S2P inactive and tether it to its substrate. morphogenesis (9). In bacteria, PDZ domain containing S2Ps often control the release of extracytoplasmic function sigma Results factors from the membrane through proteolysis of their cognate Membrane Topology of Rip1 and Its Three Anti-Sigma Factor Substrates. membrane-bound anti-sigma factors (10, 11). S2Ps are so named Many S2Ps, including E. coli RseP, are predicted to have four because the intramembrane cleavage event follows cleavage by transmembrane (TM) domains with a PDZ domain between a site-1 (S1) protease (S1P). The S1P first cleaves the periplasmic thesecondandthirdTMs(Fig.1A and ref. 25), although the domain of the anti-sigma factor in response to an extracellular reported structure of M. jannaschii S2P has six TM segments signal. Only after S1 cleavage does the S2P cleave near the cytoplasmic side of the transmembrane domain of the anti-sigma Significance factor, liberating the anti-sigma/sigma factor complex from the membrane into the cytoplasm where the sigma factor can asso- fi Site-2 proteases are ubiquitously distributed, and yet their ciate with RNA polymerase. A speci c extracytoplasmic stimulus regulation and substrate recognition remain poorly understood. can thus be coupled to a transcriptional response through a co- Here, we describe a model for site-2 protease regulation in which ordinated proteolytic cascade. regulated intramembrane protease 1 (Rip1), a site-2 protease One of the major unanswered questions in S2P-mediated RIP Mycobacterium tuberculosis fi crucial for virulence in ,istethered is how these proteases achieve speci city in signaling. In many to one of its substrates by an adapter protein, PDZ-interacting cases, S2Ps have multiple substrates, including human S2P, protease regulator (Ppr) 1. which cleaves SREBPs, ATF6, and CREB-H (12, 13); Bacillus subtilis RasP, which cleaves both RsiW as well as FtsL (14, Author contributions: J.S.S. and M.S.G. designed research; J.S.S., S.P.R., H.Y.E, and H.W.E. per- 15); and Mycobacterium tuberculosis regulated intramembrane formed research; J.S.S., S.P.R., and M.S.G. analyzed data; and J.S.S. and M.S.G. wrote the paper. protease 1 (Rip1), which cleaves three anti-sigma factors, The authors declare no conflict of interest. Anti-SigK (RskA), Anti-SigL (RslA), and Anti-SigM (RsmA) This article is a PNAS Direct Submission.M.S.W.isaguesteditorinvitedbythe (3), as well as PBP3 (16). However, there is substantial evi- Editorial Board. dence that S2Ps will cleave transmembrane segments non- 1To whom correspondence should be addressed. E-mail: [email protected]. fi speci callybothinvitroandinvivo.RsePwillcleavetheLacY This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transmembrane domain both in vitro and in vivo (17), and 1073/pnas.1305934110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1305934110 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 domain by performing a yeast two-hybrid screen of the Rip1- PDZ domain against a genome-wide M. tuberculosis protein library (28). Candidate Rip1-PDZ interactors were counterscreened against Ku, a cytoplasmic protein involved in the nonhomologous end-joining pathway of DNA repair (29, 30), in addition to the PDZ domains from PepA, PepD, and HtrA, three PDZ-containing proteases in the M. tuberculosis proteome (31). Of 7 × 104 candidate fusions screened, we identified four specific Rip1 PDZ interactors after counter screening. Two of these activation domain (AD) fusions contained fusions to the uncharacterized proteins encoded by rv3333c and rv3439c, which we named Ppr1 and Ppr2, respectively. Both Ppr1 and Ppr2 interact with the Fig. 1. Membrane topology of Rip1 and its three anti-sigma factor sub- Rip1 PDZ, but not with Ku or the PDZ domains from PepA, strates. (A) Schematic representation of M. tuberculosis Rip1 illustrating the PepD, or HtrA (Fig. 2A). Ppr1 and Ppr2 are both proline- and four predicted transmembrane domains (TM1 through -4, in gray) and the alanine-rich proteins that share significant sequence similarity in PDZ domain between TM2 and 3 (in green). The vertical lines marked with their C-terminal regions (Fig. S1B). The M. tuberculosis yeast numbers 1–5 mark the fusion points to either β-galactosidase or alkaline two-hybrid library was constructed from partial digests of geno- phosphatase as follows: 1, Y89; 2, V145; 3, W218; 4, G361; and 5, L383. (B) mic DNA and therefore the AD-Ppr1 fusions identified in the Membrane topology of Rip1. lacZ (Upper) and phoA (Lower) fusions to Rip1 screen encoded truncated versions of the Ppr1 protein, all of at the positions indicated in A were expressed in M. smegmatis along with a vector control (vect) or a positive control (+)forlacZ (consisting of unfused which included the C terminus of Ppr1 beginning at amino acid lacZ)orphoA [an antigen85-phoA fusion (26)]. The upper image shows cells 131. The Ppr1 protein contains a predicted transmembrane do- bearing each β-galactosidase fusion cultured on media containing X-gal and main at its N terminus (amino acids 6–25) but has no identifiable the bottom panel shows each PhoA fusion cultured on media containing enzymatic function or conserved domains. BLAST searching of BCIP. (C) Membrane topology of Rip1 substrates. β-Gal or PhoA was fused to the M. tuberculosis Ppr1 protein sequence identified Ppr1 in the C terminus of Anti-SigK (RskA), Anti-SigL (RslA), or Anti-SigM (RsmA), pathogenic slow-growing mycobacteria, including Mycobacterium and the plasmids encoding these fusions were transformed into M. smegmatis bovis, Mycobacterium africanum, and Mycobacterium marinum, and cultured on media containing X-gal (Upper)orBCIP(Lower). The positive which contains three Ppr1 paralogs (Fig. S1A). We did not controls for X-gal and BCIP are the same as used in B.(D) Experimentally determined topology of Rip1 and its substrates. identify Ppr1 orthologs in M. smegmatis or in any other bacterial taxa outside of pathogenic mycobacteria. M. smegmatis expressing a PhoA fusion to the C terminus of Ppr1 had PhoA activity, (18). However, the topology of a PDZ-containing S2P has not indicating localization of the C terminus of Ppr1 after amino acid been directly examined experimentally. We investigated the to- 70 to the extracytoplasmic space (Fig. S1C). pology of M. tuberculosis Rip1 in the mycobacterial membrane by constructing enzymatic fusions to alkaline phosphatase (encoded by phoA)andβ-galactosidase (encoded by lacZ). Alkaline phosphatase is active in the periplasm and produces a blue colony in the presence of BCIP, whereas β-galactosidase is active in the cytoplasm and produces a blue colony in the presence of X-gal (26). Mycobacterium smegmatis expressing fusions to phoA or lacZ between TM1 and TM2 or TM3 and TM4 (fusions 1 and 4, respectively, in Fig. 1A)demonstratedβ-gal activity, but not PhoA activity, consistent with localization to the cytoplasm (Fig. 1B). Conversely, two fusions within the PDZ domain (amino acids 124– 230) at residues 145 and 218 exhibited PhoA activity, but not β-galactosidase activity, indicating localization to the periplasm (fusions 2 and 3, respectively; Fig. 1B). Thus, expression of these fusions in M. smegmatis indicated that the PDZ domain of Rip1 is extracytoplasmic. We next determined the membrane topology of the three identified Rip1 substrates, Anti-SigK, Anti-SigL, and Anti-SigM (3). M. smegmatis expressing phoA or lacZ fusions of the C ter- Fig. 2. Ppr1 is a specific interactor of the Rip1-PDZ domain. (A)EGY48 mini of these anti-sigma factors were blue on agar containing yeast cells containing plasmids encoding AD fusions to Ppr1 (encoded by BCIP, but not X-gal, indicating localization of the C termini to M. tuberculosis Rv3333c) or Ppr2 (encoded by M. tuberculosis Rv3439c)and the periplasm (Fig. 1C). Taken together, these data indicate that a LexA-LacZ reporter plasmid were tested with LexA-DNA BD fusions to Rip1- the Rip1-PDZ domain and the C termini of Rip1 substrates PDZ, the NHEJ protein Ku, PepA-PDZ, PepD-PDZ, or HtrA-PDZ. The Ppr1 and Ppr2 AD fusions are under the control of a galactose-inducible promoter, colocalize in the extracytoplasmic compartment (Fig. 1D). which is repressed by glucose. (B)Specificity of the Ppr1-Rip1-PDZ interaction. Ppr1-Myc was coexpressed in E. coli with either Rip1-PDZ-His, PepA-PDZ-His, or Protein Interactors of the Rip1-PDZ Domain. The PDZ domain is HtrA-PDZ-His. Soluble lysates from the indicated combinations of expressed a widely distributed protein–protein interaction motif that often proteins or vector controls were subjected to metal-affinity chromatography organizes signaling complexes through binding C-terminal pep- to purify histidine tagged proteins. Supernatants (sn) and eluted proteins (e) tides (24). Deletion of the PDZ domain of E. coli RseP causes were analyzed by immunoblotting with anti-Myc or anti-His antibodies. The hyperactivation of the SigE pathway, DegS (S1P), independent lanes labeled “-” are no-isopropyl β-D-1-thiogalactopyranoside (IPTG) control “ ” cleavage of RseA, and insensitivity of SigE activation to RseB samples, and the lanes labeled x are blank lanes. (C) Ppr1 and Rip1-PDZ interact in vitro. Ppr1 lacking its transmembrane domain (Ppr1-no TM; amino inhibition (20, 21, 23, 27). However, no proteins have been – fi fi acids 32 281) and either His-SUMO or His-SUMO-Rip1-PDZ were mixed and identi ed that interact with the RseP-PDZ speci cally, or S2P- purified by metal-affinity chromatography. Bound proteins were eluted with PDZs more broadly, that may mediate these effects. We screened imidazole, and the input protein (I), wash (W), and elution (E) were separated for M. tuberculosis proteins that interact directly with the Rip1-PDZ by SDS/PAGE and proteins visualized by staining with Coomassie blue.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1305934110 Schneider et al. Downloaded by guest on September 30, 2021 by complementation of the Δppr1 strain with a WT copy of ppr1 (Fig. 3B and Fig. S3D). Conversely, overexpression of Ppr1 repressed rpfC levels in WT M. tuberculosis but had no effect on rpfC levels in the Δrip1 strain (Fig. 3B). Thus, loss of Ppr1 phenocopies loss of the Rip1-PDZ and overexpression of Ppr1 phenocopies loss of Rip1, consistent with Ppr1 repressing the Rip1 pathway through the Rip1-PDZ.

Ppr1 Scaffolds Rip1 to an Anti-Sigma Factor Substrate via the PDZ Domain. The data above indicate that Ppr1 interacts directly with Rip1-PDZ in the periplasmic space and that the C termini of the three known anti-sigma factor substrates also localize to this Fig. 3. Loss of Ppr1 hyperactivates the Rip1 pathway. (A) C57Bl6 mice were compartment. In E. coli, the RseB protein interacts directly with infected by aerosol with either WT M. tuberculosis Erdman (WT, inverted tri- the C terminus of RseA and negatively regulates the SigE angle), or M. tuberculosis Δppr1::hyg (circle). Bacterial titers in the lung were pathway (32, 33). To understand the protein interactors of the C determined over time with 3–5 mice per group at each time point. (B)Roleof termini of the three Rip1 anti-sigma factor substrates, we per- Ppr1 and the Rip1-PDZ in Rip1 pathway activation. Quantitative RT-PCR was formed yeast two-hybrid screens using the C termini of these used to measure the abundance of the mRNA encoding RpfC in the indicated anti-sigma factors as baits. Surprisingly, this screen identified M. tuberculosis strains. Error bars represent the SEM. Strains are WT, WT car- fi rying a plasmid directing the expression of Rip1 lacking the PDZ domain (WT+ Ppr1 as a speci c interactor with the extracytoplasmic domain of Rip1ΔPDZ, MGM575), Δrip1(MGM350), Δrip1+ aWTcopyofrip1 (MGM350), RsmA (RsmA-C), but not RskA (Anti-SigK) (Fig. 4A). The Δppr1 (JSS001), Δppr1 complemented strain (Δppr1+ppr1, JSS002), Δrip1 with RsmA-C interacting Ppr1 fragments isolated from the two- ppr1 (Δrip1+ppr1, JSS004), and WT with ppr1 (WT+ppr1, JSS003). hybrid screen included Ppr1 amino acids 59–281 (Fig. 4A), suggesting that RsmA may interact with a distinct region of Ppr1 fi To confirm the specificity of Ppr1 for the Rip1-PDZ domain, from that of Rip1-PDZ. To de ne the interacting regions of Ppr1 we performed copurification experiments with Ppr1 (30-281)- with Rip-PDZ and RsmA-C, we constructed serial truncations of Myc and His-tagged PDZ domains from M. tuberculosis expressed Ppr1 and tested them for interaction with Rip1-PDZ, RsmA, and fi RskA in the yeast two-hybrid system. Ppr1 (59-281) and Ppr1

in E. coli. Ppr1(30-281)-Myc copuri ed with Rip1-PDZ-His, but MICROBIOLOGY not with PepA-PDZ-His nor HtrA-PDZ-His (Fig. 2B). Taken (73-281) interacted with both RsmA and Rip1-PDZ, but not together, these experiments confirm that Ppr1 interacts specifically with the PDZ domain of Rip1 but not the PDZ domains from other membrane-embedded proteases from M. tuberculosis. To confirm the interaction of full-length Ppr1 with the Rip1- PDZ domain in vitro, we purified the Rip1-PDZ as a His-SUMO fusion protein and Ppr1 lacking its N-terminal transmembrane domain (residues 30–281). Nickel agarose affinity chromatography of mixtures of His-SUMO-Rip1 PDZ and Ppr1 (30-281) copurified Ppr1, whereas a control His-SUMO protein did not (Fig. 2C). These experiments confirm that Ppr1 interacts directly with the PDZ domain of Rip1.

Ppr1 Suppresses the Rip1 Pathway but Does Not Affect Virulence. To explore the function of ppr1 in the Rip1 pathway, we constructed a deletion mutant of ppr1 in M. tuberculosis. Through Southern blot analysis, we confirmed deletion of ppr1 and thus demonstrated that ppr1 is not essential (Fig. S2). Because rip1 is critical for M. tuberculosis virulence (4) and Ppr1 interacts with the Rip1-PDZ domain, we assessed the role of ppr1 on virulence Δ using the mouse model of aerosol infection. M. tuberculosis ppr1 Fig. 4. Ppr1 interacts with both Rip1-PDZ and Anti-SigM. (A) Interaction of displayed similar growth kinetics to wild type (WT), as measured Ppr1 with RsmA and Rip1-PDZ. Yeast strains carrying a LexA-LacZ reporter by lung colony-forming units in the first three weeks of infection plasmid and plasmids encoding AD fusions to Ppr1 with the amino acid (Fig. 3A). There was also no significant difference between titers boundaries listed above each column and LexA-DNA BD fusion to Rip1-PDZ, of WT M. tuberculosis and Δppr1 during the chronic phase of in- RsmA-C, or RskA-C. The strains are shown on X-gal–containing media with fection (Fig. 3A). Because the Δrip1 strain is attenuated in both either glucose (Upper) or galactose (Lower), which induces expression of the AD fusion proteins. (B) Mixtures of His-SUMO-RslA-C/Ppr1 (Left) or His- acute and chronic infection (4), the lack of substantial virulence fi fi Δppr1 ppr1 SUMO-RsmA-C/Ppr1 (Right) were puri ed by metal-af nity chromatography. phenotype of the strain indicates that is not an es- Bound proteins were eluted with imidazole, and the input protein (I), wash sential cofactor for all Rip1 functions. (W), and elution (E) were separated by SDS/PAGE and proteins visualized by We next explored direct effects of ppr1 on the Rip1 pathway in staining with Coomassie blue. (C) Mixtures of Ppr1/His-SUMO/RsmA-C or vivo through analysis of the mRNA for rpfC, a gene positively Ppr1/His-SUMO-Rip1-PDZ-/RsmA-C were purified by metal-affinity chroma- regulated by the Rip1 pathway (3, 4). As previously reported, tography. Bound proteins were eluted with imidazole, and the input protein rpfC mRNA is underexpressed in the Δrip1 strain and restored in (I), wash (W), and elution (E) were separated by SDS/PAGE and proteins the Δrip1 strain complemented by a WT copy of rip1 (ref. 3 and visualized by staining with Coomassie blue. (D) Specificity of the Ppr1-RsmA Fig. 3B). WT M. tuberculosis expressing Rip1 lacking its PDZ interaction. Ppr1-Myc was coexpressed in E. coli with the C-terminal frag- domain (Rip1-ΔPDZ) overexpressed rpfC, consistent with a hy- ments of RsmA-His, RskA-His, or RslA-His. Soluble lysates from the indicated combinations of expressed proteins or vector controls were subjected to peractive Rip1 pathway conferred by loss of the Rip1 PDZ do- metal-affinity chromatography to purify histidine-tagged proteins. Super- main (Fig. 3B). Loss of ppr1 resulted in increased rpfC mRNA natants (sn) and eluted proteins (e) were analyzed by immunoblotting with levels similar to the overexpression observed in M. tuberculosis anti-Myc or anti-His antibodies. The lanes labeled “-” are no-IPTG control expressing Rip1ΔPDZ (Fig. 3B), a phenotype that was reversed samples, and the lanes labeled “x” are blank lanes.

Schneider et al. PNAS Early Edition | 3of6 Downloaded by guest on September 30, 2021 Fig. 5. Ppr1 accelerates Rip1 cleavage of S1-cleaved RsmA. (A) Experimental Schematic. ATC induces expression of HA-Ppr1. A control strain carries an ATC- inducible vector without Ppr1 sequences. At time −30 min, cells are labeled with SNAP-505, which labels SNAP-tagged proteins covalently, for 30 min. SNAP-

505 labeling is terminated by addition of O6 benzylguanine (O6-BG). Aliquots are taken at time 0, 60 min, and 180 min, and protein lysates are prepared. The right side of A depicts the full-length RsmA-SNAP, full-length RsmA-SNAP 505, and S1P-cleaved RsmA-SNAP 505, which is the substrate for Rip1. (B) Kinetics of RsmA degradation with and without Ppr1. Protein lysates from WT M. smegmatis expressing RsmA-SNAP and either vector (JSS182) (left blots) or ATC- inducible Ppr1 (JSS183, right blots) were prepared at the indicated time points and separated by SDS/PAGE. Fluorescently labeled RsmA-SNAP was detected by gel ﬂuorometric scanning (upper blots). The positions of full-length RsmA-SNAP, S1P-cleaved RsmA-SNAP (S1) and the S2P-cleaved product (S2P) are indicated at the side of the gel image. Ppr1 protein was detected by immunoblotting with anti-HA antibodies and protein loading was normalized using antibodies to CarD. (C) The normalized fraction of RsmA-S1 over time in vector –ATC (red), vector +ATC (black), Ppr1 –ATC (green), and Ppr1 +ATC (blue). Error bars are SEM, and each point is the mean of four independent experiments. The bar graph depicts data from the 180-min time point. (D) The accelerated degradation of RsmA-S1 by Ppr1 is dependent on Rip1. The experimental design is identical to B, but the parent strain is M. smegmatis Δrip1.

RskA (Fig. 4A). Ppr1 (87-281) failed to interact with RsmA, but Rip1 to RsmA while simultaneously inhibiting Rip1 cleavage retained interaction with Rip1-PDZ. Deletion of the C-terminal through the Rip1 PDZ domain, thereby inhibiting RsmA cleav- 33 amino acids of Ppr1 did not affect interaction with Rip1-PDZ, age. With SigM pathway activation, Ppr1 might facilitate Rip1 indicating that, in contrast to many ligand-PDZ interactions, the mediated proteolysis of S1P-cleaved RsmA by tethering Rip1 to Ppr1-Rip1PDZ interaction does not require the C terminus of RsmA. Loss of Ppr1 leads to broad activation of the Rip1 Ppr1. Truncation of Ppr1 beyond amino acid 248 from the C pathway that includes both SigM-dependent (Fig. S3) and SigM- terminus abolished interaction with Rip1-PDZ (Fig. 4A). independent genes (Fig. 3B and Fig. S3), indicating that one fi To further con rm the direct interaction between Rip1-PDZ, effect of Ppr1 is to generally prevent Rip1 activation. To test the Ppr1, and RsmA, we purified Rip-PDZ, Ppr1, and the C-termi- fi fi effect of Ppr1 on RsmA cleavage, we sought to quantify the half- nal fragment of RsmA (RsmA-C). Af nity puri cation of His- lives of the cleavage products of RsmA in vivo. In vitro re- SUMO-RsmA coprecipitated Ppr1, whereas His-SUMO-RslA constitution of S1P/S2P systems has been difficult and our prior did not (Fig. 4B). When all three proteins were mixed, affinity efforts to identify the S1Ps in the Rip1 pathway have been purification of His-SUMO-Rip1-PDZ copurified both Ppr1 E. coli and RsmA-C, whereas a control His-SUMO protein did not unsuccessful (3), precluding in vitro analysis. In WT (6), purify either RsmA-C or Ppr1 (Fig. 4C). We further confirmed B. subtilis (36), and M. tuberculosis (3), the S1P-cleaved anti- the specificity of the Ppr1-RsmA interaction by coexpression sigma factor intermediate is very short-lived and not detectable in E. coli. When coexpressed with anti-sigma factor C termini in WT cells. In cells lacking the S2P, this intermediate accu- in E. coli, Ppr1 only copurified with RsmA-C but not RslA-C mulates. To enhance our ability to quantify the S1-cleaved anti- or RskA-C (Fig. 4D). These experiments indicate that the Rip1- sigma factor intermediates in WT cells, we adapted the O6- PDZ and RsmA both interact with Ppr1, RsmA at the Ppr1 N alkylguanine-DNA-alkyltransferases (hAGT, SNAP tag) protein terminus and Rip1-PDZ through the Ppr1 C terminus. These fusion system (37, 38) for mycobacteria, which allows covalent interactions are exclusive to RsmA and do not extend to the two labeling of fusion proteins in vivo. We constructed N-terminal other known anti-sigma factor substrates of Rip1, RskA and SNAP-RsmA and SNAP-RslA fusions. Preliminary experiments RslA, indicating that Ppr1 is a substrate-specific adapter protein confirmed that the SNAP fusions were expressed in M. smegmatis, that bridges between Rip1 and one of its substrates. were labeled efficiently by SNAP-505 (a green fluorescent O6 benzylguanine derivative), and labeling was quenched by un- Ppr1 Modulates the Sigma Factor M Branch of the Rip1 Pathway. labeled O6 benzylguanine (O6-BG). We expressed SNAP-RsmA Based on the interaction of Ppr1 with both Rip1-PDZ and RsmA, we hypothesized that Ppr1 may affect activation of the SigM branch of the Rip1 pathway. SigM positively auto-regulates its own transcription (34), and therefore we measured the abundance of the mRNA encoding SigM by two methods, quantitative RT-PCR and NanoString (35), in the Δppr1 strain. By both methods, we observed that the mRNA encoding SigM was overexpressed in Δppr1 cells compared with either WT or Δppr1 complemented cells (Fig. S3 A–C). We were unable to detect an effect of ppr1 deletion on the SigK or SigL pathways, but these fi experiments were hampered by the low expression levels of these Fig. 6. Adapter protein model of Rip1 substrate speci city. Ppr1 binds to both the Rip1 PDZ domain and the C terminus of the RsmA Rip1 substrate. regulons and the unknown inducing signals of these pathways. Rip1 is tethered to RsmA by Ppr1 through the Rip1-PDZ, thereby holding Rip1 inactive. S1 cleavage of RsmA, which is not inhibited by Ppr1, releases Ppr1 Accelerates Rip1 Cleavage of S1P-Cleaved RsmA but Not RslA. the inhibitory effect of Ppr1 on the PDZ domain, allowing Rip1 cleavage. The The data above suggest that Ppr1 may specifically control the model predicts additional unidentified substrate-specific adapters that interaction of Rip1 with RsmA. One model is that Ppr1 tethers tether Rip1 to Anti-SigK and Anti-SigL.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1305934110 Schneider et al. Downloaded by guest on September 30, 2021 or SNAP-RslA in M. smegmatis carrying a tetracycline inducible alternative model to understand the role of the S2P PDZ domain HA-Ppr1, allowing us to temporally control Ppr1 expression. in coupling and S2P self-inhibition. By binding the PDZ at its C The experimental scheme is depicted in Fig. 5A. We induced terminus and RsmA at its N terminus, Ppr1 is able to simulta- Ppr1 expression with anhydrotetracycline (ATC) 45 min before neously tether Rip1 to its substrate, while also holding Rip1 in- labeling SNAP-RsmA with SNAP-505 for 30 min. Labeling was active (Fig. 6). When the S1P cleaves RsmA, the presence of terminated by addition of unlabeled O6-BG and samples col- Ppr1 accelerates Rip1 cleavage, consistent with a role in coupling lected over a 180-min time course for analysis. Cells carrying the the two proteolytic events. ATC-inducible vector served as controls for any effects of ATC We also propose that Ppr1 provides a model for understanding treatment on RsmA stability. The intensities of RsmA-SNAP-505 the substrate selection of S2Ps, which is poorly understood. Al- fluorescent species were normalized to total protein abundance though amino acid residues have been identified in cleaved TM by immunoblotting for CarD (39). We detected three fluorescent segments that affect iCLIP cleavage, these residues affect cleavage protein species of SNAP-RsmA, corresponding to full-length of both physiologic and nonphysiologic substrates (44, 45) and (full), S1-cleaved (S1), and final site-2–cleaved (Fig. S2 and a conserved S2P cleavage motif has not been identified. Multiple Fig. 5B). The S1 product was observed as two closely migrating studies have demonstrated that S2Ps will cleave transmembrane species, consistent with prior data showing anti-sigma factor segments of proteins that are not natural substrates (17, 18), trimming by multiple proteases (40). Over the 180-min time strongly suggesting that additional determinants must limit S2P course, the half-life of S1-cleaved RsmA was unaffected by ATC cleavage to physiologic substrates while excluding other trans- treatment in vector bearing control cells (Fig. 5 B and C). In membrane proteins. We propose that Ppr1 is a trans-acting factor contrast, expression of Ppr1 modestly accelerated the clearance that provides S2P specificity by tethering Rip1 to one substrate of RsmA-S1 (P = 0.01 for comparison between Ppr1 and Ppr1 (among three that have been identified), thereby facilitating RsmA plus ATC at the 180-min time point; Fig. 5C). The half-life of cleavage while inhibiting nonspecific cleavage. This model implies fi RsmA-S1 was signi cantly shorter in the presence of Ppr1 (117 that there may be additional adapter proteins which function = min with Ppr1 compared with 181 min in control cells; P 0.025 analogously to Ppr1 in tethering Rip1 to its other anti-sigma factor for comparison of half-lives). We did not observe a consistent or substrates, and leading candidates are the additional Ppr proteins fi signi cant difference in the rate of clearance of full-length that we have identified as binding the Rip1-PDZ. We also speculate RsmA-SNAP with Ppr1 expression, indicating that Ppr1 specif- that the adapter protein model of S2P-substrate bridging could ically affects clearance of the S1-cleaved RsmA. apply to other PDZ containing S2Ps outside of mycobacteria for

fi MICROBIOLOGY To con rm that Rip1 mediates the acceleration of S1 clear- which PDZ-interacting proteins have yet to be identified. ance with Ppr1 expression, we quantitated the clearance of the S1 intermediate in Δrip1 cells with or without Ppr1. As previously Materials and Methods Δ reported (3), the RsmA-S1 intermediate accumulates in rip1 Bacterial Strains and Growth Conditions. All bacterial strains for this study are cells (Fig. 5D). In the absence of Rip1, Ppr1 did not accelerate listed in Table S1. M. tuberculosis strains (all based on the WT strain Erdman the clearance of RsmA-S1 in comparison with control cells (Fig. EG1, which is animal-passaged) were grown at 37 °C in 7H9 (broth) or 7H10 5D). To determine whether the effect of Ppr1 on RsmA-S1 (agar) (Difco) media with oleic acid, albumin, dextrose, catalase (OADC) clearance was specific for RsmA, we measured the clearance of enrichment, 0.5% glycerol, and 0.05% Tween 80 (broth media only). M. SNAP-RslA with and without Ppr1 using the same experimental smegmatis strains were cultured at 37 °C on Luria–Bertani (LB) medium design (with an additional longer induction of Ppr1 of 6 h; Fig. containing 0.5% dextrose, 0.5% glycerol, and 0.05% Tween 80 (broth). When appropriate, the following were added into growth medium for E. S4). We did not observe any effect of Ppr1 on clearance of − coli/Mycobacteria, respectively: chloramphenicol (Sigma) at 25 μg·mL 1, SNAP-RslA-S1 (Fig. S4), indicating that the effect of Ppr1 on S1 μ · −1 fi hygromycin B (Boehringer Mannheim) at 150/50 g mL , kanamycin (Sigma) clearance is speci c for RsmA. These data indicate that Ppr1 at 40/20 μg·mL−1, streptomycin (MP Biomedicals) at 40/20 μg·mL−1,ATC fi −1 speci cally accelerates Rip1 cleavage of the S1-cleaved RsmA, (Sigma) at 50 ng·mL , 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside − but not other Rip1 substrates. (X-gal) (Fisher Scientific) at 50 μg·mL 1, 5-bromo-4-chloro-3-indolyl phos- phate (BCIP) (Sigma) at 60 μg·mL−1. Discussion Despite the wide phylogenetic distribution of S2Ps and diverse Yeast Strains and Growth Conditions. All yeast strains for this study are listed in biologic functions, their regulation remains poorly understood. Table S2.SeeSI Materials and Methods for yeast growth conditions. All yeast In most cases, S2P-mediated signaling cascades follow the same and bacterial plasmids are listed in Table S3. general paradigm: (i)aS1Pfirst cleaves the extracytoplasmic domain of a transmembrane substrate in response to a specific Yeast Two-Hybrid Screen for M. tuberculosis Rip1-PDZ–Binding Proteins. The inducing signal [e.g., unfolded OMPs in the case of the S1P bait plasmid comprised a fusion of the LexA DNA-binding domain (BD) (DegS) in the E. coli SigE pathway] upon which (ii) S2P cleavage encoded in pEG202 to the N terminus of the Rip1 PDZ domain. Character- ization of the bait fusion protein and the interaction screen against an within the transmembrane segment of the substrate rapidly ensues, M. tuberculosis activation domain (AD) fusion library were performed as thereby liberating a cytosolic fragment of the substrate. This S1P/ previously described (28). DNA-BD fusions to the N terminus of Ku, HtrA- S2P coupling necessitates that the S2P remain inactive until the PDZ, PepA-PDZ, and PepD-PDZ were similarly constructed and characterized S1P cleavage has occurred, a coupling event that is not well for use in the yeast two-hybrid system (see strain plasmid table for specific understood. Observations in prokaryotic systems suggest that the amino acid boundaries). Expression of full-length LexA-DNA BD bait fusions PDZ domains of S2Ps participate in this S1P-S2P coupling be- and AD–prey fusions was confirmed by immunoblotting with anti-LexA and cause this domain is necessary to hold the protease inactive until anti-HA antibodies, respectively. site one proteolysis occurs (20, 21, 23, 27, 41). Recent structural data suggested that one function of the RseP PDZ domain is to Protein Purification, Coexpression, Copurification, and in Vitro Protein Interactions. bind the cleaved C terminus of the RseA anti-sigma factor See SI Materials and Methods for details. generated by DegS (42), potentially providing a mechanism of ppr1 rv3333c M. tuberculosis Δ coupling. However, recent in vivo examination of this model did Deletion of ( ) from . M. tuberculosis ppr1 was constructed via specialized transduction using the temperature-sensitive not support this mechanism (27). Other recent data indicate that phage phAE87 as previously described (46). ppr1 was replaced with a acid can activate RseA cleavage by RseP independent of DegS in hygromycin resistance cassette by deleting all but the start and stop S. typhimurium (41) and that LPS biosynthetic intermediates are codons of ppr1. The Δppr1 allele was verified by Southern blotting using the a second signal for RseA degradation through binding RseB 3′ flanking region of ppr1 as a probe of chromosomal DNA fragmented with (43). We propose that Ppr1 in M. tuberculosis provides an NcoI. The predicted size for WT is 884 bp, and the Δppr1 allele is 3,431 bp.

Schneider et al. PNAS Early Edition | 5of6 Downloaded by guest on September 30, 2021 Quantitative RT-PCR. mRNA levels were measured with quantitative RT-PCR as performed with ImageJ by calculating the area under the curves measuring previously described (3). See SI Materials and Methods for additional details. band intensity. The fluorescence intensity of SNAP-tagged proteins was Oligonucleotides are listed in Table S4. adjusted to the intensity of the loading control CarD for the same protein sample. For analysis of the S1-cleaved intermediates of RsmA and RslA, we SNAP Pulse-Chase Analysis. Cultures were grown in LB medium with appro- quantitated the sum of both intermediates and each intermediate in- ∼ priate antibiotics to an OD600 of 0.6, induced with ATC for the time in- dependently, with similar results. Data shown are for the sum of the μ dicated, and then pulsed with 1 M SNAP-Cell 505 reagent (NEB) for 30 min. two intermediates. Cells were then collected by centrifugation and resuspended in fresh LB with 6 1mMO-benzylguanine (Sigma) in DMSO (a concentration determined in Murine Infection. Murine experiments were performed as previously de- preliminary experiments to block further labeling); 1-mL aliquots from the scribed (39) and in accordance with National Institutes of Health guidelines time points indicated were snap-frozen. Cells were lysed at 37 °C in Tri- and for housing and care of laboratory animals, and were approved by the s·EDTA with 10 mg·mL−1 lysozyme for 45 min, incubated for 10 min at 100 °C Memorial Sloan-Kettering Institutional Animal Care and Use Committee in SDS/PAGE loading buffer. Proteins were resolved on NuPAGE 4–12% Bis- (IACUC). Tris polyacrylamide gels (Invitrogen). Fluorescently labeled SNAP proteins were detected on a Typhoon Trio (GE Healthcare) using the 526-nm Fluo- rescein emission filter and Green (532-nm) laser, with the photomultiplier ACKNOWLEDGMENTS. We thank Miriam Braunstein for providing phoA fusion vectors and Allison Fay for insights into use of the SNAP tag. This set to 450, and visualized with ImageQuant (GE Healthcare). The same gels work was supported by National Institutes of Health (NIH) Grant AI53417 used for SNAP detection were transferred to nitrocellulose membranes and (to M.S.G.). J.S.S. is supported by NIH Grant T32 AI007621 (Weill Cornell probed with anti-HA antibody (HA.11 Clone 16B12; Covance) to detect Graduate School of Medical Sciences) and the Dorris J. Hutchison Fellowship HA-Ppr1, and monoclonal anti-CarD as a loading control. Quantification (Sloan-Kettering Institute). H.W.E. is supported by the Gerstner Sloan Ketter- of Western blots and fluorescently labeled SNAP-tagged proteins was ing Summer Undergraduate Research Program.

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