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

Abstract

Site-2 proteases (S2Ps) are intramembrane metalloproteases that cleave transmembrane substrates in all domains of life. Many S2Ps, including human S2P and Mycobacterium tuberculosis Rip1, have multiple substrates in vivo, which are often transcriptional regulators. However, S2Ps will also cleave transmembrane sequences of nonsubstrate proteins, suggesting additional specificity determinants. Many S2Ps also contain a PDZ domain, the function of which is poorly understood. Here, we identify an M. tuberculosis protein, PDZ-interacting protease regulator 1 (Ppr1), which bridges between the Rip1 PDZ domain and anti-sigma factor M (Anti-SigM), a Rip1 substrate, but not Anti-SigK or Anti-SigL, also Rip1 substrates. In vivo analyses of Ppr1 function indicate that it prevents nonspecific activation of the Rip1 pathway while coupling Rip1 cleavage of Anti-SigM, but not Anti-SigL, to site-1 proteolysis. Our results support a model of S2P substrate specificity in which a substratespecific adapter protein tethers the S2P to its substrate while holding the protease inactive through its PDZ domain.intramembrane proteolysis | signal transduction | Tuberculosis | Sigma factor R egulated intramembrane proteolysis (RIP) is a widely distributed mechanism of signal transduction across membranes in which specialized intramembrane cleaving proteases (iCLIPs) cleave the transmembrane segments of substrate proteins (1, 2). The site-2 (S2) proteases (S2Ps) are one class of iCLIP that is widely distributed from bacteria to human cells and participate in such diverse pathways as lipid biosynthesis (1, 3, 4), sporulation (5), membrane stress (6, 7), alginate production (8), and polar morphogenesis (9). In bacteria, PDZ domain containing S2Ps often control the release of extracytoplasmic function sigma factors from the membrane through proteolysis of their cognate membrane-bound anti-sigma factors (10, 11). S2Ps are so named because the intramembrane cleavage event follows cleavage by a site-1 (S1) protease (S1P). The S1P first cleaves the periplasmic domain of the anti-sigma factor in response to an extracellular signal. Only after S1 cleavage does the S2P cleave near the cytoplasmic side of the transmembrane domain of the anti-sigma factor, liberating the anti-sigma/sigma factor complex from the membrane into the cytoplasm where the sigma factor can associate with RNA polymerase. A specific extracytoplasmic stimulus can thus be coupled to a transcriptional response through a coordinated proteolytic cascade.One of the major unanswered questions in S2P-mediated RIP is how these proteases achieve specificity in signaling. In many cases, S2Ps have multiple substrates, including human S2P, which cleaves SREBPs, ATF6, and CREB-H (12, 13); Bacillus subtilis RasP, which cleaves both RsiW as well as FtsL (14, 15); and Mycobacterium tuberculosis regulated intramembrane protease 1 (Rip1), which cleaves three anti-sigma factors, Anti-SigK (RskA), Anti-SigL (RslA), and Anti-SigM (RsmA) (3), as well as PBP3 (16). However, there is substantial evidence t...

The Rip1 Protease of Mycobacterium tuberculosis Controls the SigD Regulon

“…Though ECF sigma factors are regulated by extracytoplasmic stimuli, only some are held inactive by a cognate membrane-embedded anti-sigma factor which would be a candidate substrate for Rip1. We previously demonstrated the topology of anti-sigma factors K, L, and M as transmembrane proteins with extracytoplasmic C termini (15). To search for additional anti-sigma factors that might be candidate Rip1 substrates, we analyzed additional anti-sigma factors annotated in the M. tuberculosis genome for putative transmembrane domains and determined their topology in the mycobacterial membrane.…”

“…Fusions of the C termini of RsdA and RsgA to alkaline phosphatase (PhoA) or ␤-galactosidase (␤-Gal) were constructed by ablating the termination codon of RsdA or RsgA such that the terminal amino acid (H299 for RsdA or T244 for RsgA) was fused to the coding sequence of either lacZ or phoA. Alkaline phosphatase and ␤-galactosidase assays were performed as previously described (15).…”

Themes and variations in gene regulation by extracytoplasmic function (ECF) sigma factors

“…The initiating protease(s) has not yet been identified. Recently it was shown that an adaptor protein, Ppr1, binds to both RsmA and the substrate binding domain of Rip1 and prevents signal-independent degradation of RsmA (Figure 4c) [60]. The adaptor is proposed to provide specificity to the system by tethering RsmA to the protease.…”

“…The adaptor is proposed to provide specificity to the system by tethering RsmA to the protease. This model predicts that additional adaptor proteins exist to connect Rip1 with each of the remaining anti-sigma factors [60]. …”

Discrimination and Integration of Stress Signals by Pathogenic Bacteria

“…The mycobacterial Rip1 protease can degrade anti-sigma factors of σ M , σ K and σ L . Adaptor proteins may help Rip1 achieve specificity in signaling, as suggested by selective tethering of the Rip1 PDZ domain to the anti-sigma factor σ M , but not to the anti-sigma factors σ K and σ L , by the adaptor Ppr1 (Schneider et al, 2013). Allosteric interactions between the adaptor SafA and the PhoQ periplasmic sensor domain specifically link EvgS/EvgA and PhoQ/PhoP signaling in response to acid pH (Eguchi et al, 2012).…”