RseP (YaeL), an Escherichia coli RIP protease, cleaves transmembrane sequences

Abstract

Escherichia coli RseP (formerly YaeL) is believed to function as a 'regulated intramembrane proteolysis' (RIP) protease that introduces the second cleavage into anti-sigma(E) protein RseA at a position within or close to the transmembrane segment. However, neither its enzymatic activity nor the substrate cleavage position has been established. Here, we show that RseP-dependent cleavage indeed occurs within predicted transmembrane sequences of membrane proteins in vivo. Moreover, RseP catalyzed the same specificity proteolysis in an in vitro reaction system using purified components. Our in vivo and in vitro results show that RseP can cleave transmembrane sequences of some model membrane proteins that are unrelated to RseA, provided that the transmembrane region contains residues of low helical propensity. These results show that RseP has potential ability to cut a broad range of membrane protein sequences. Intriguingly, it is nevertheless recruited to the sigma(E) stress-response cascade as a specific player of RIP.

Figure 1

Proteolytic substrates of RseP used in this study. (A) Schematic representations of RseA derivatives and other RseP substrates. Regions having RseA-derived sequence are shown in white, MBP in black, LacY-derived sequence in dark gray and Bla-derived sequence striped. (B) The amino-acid sequences of the TM region of RseA (RseATM), the first (LacYTM1) and the fifth (LacYTM5) TM region of LacY and the signal peptide of Bla (SS^Bla) used in the above constructs.

Figure 2

RseP-dependent cleavage of RseATM, LacYTM1 and LacYTM5 having N-terminal HA and MBP. (A) Immunological detection of the cleaved N-terminal products. Plasmids pSTD797 (encoding HA-MBP-RseA140; RseATM), pSTD835 (HA-MBP-RseA(LacYTM1)140; LacYTM1) and pSTD843 (HA-MBP-RseA(LacYTM5)140; LacYTM5) were introduced into a ΔrseA strain (AD1811, lanes 1, 5 and 9), as well as into its derivatives additionally deleted for rseP (KK211, lanes 2, 6 and 10), degS (AD1839, lanes 3, 7 and 11) or both (AD1840, lanes 4, 8 and 12). Plasmid-bearing cells were grown in L broth containing 1 mM IPTG and 1 mM cAMP at 30°C. Proteins were analyzed by SDS–PAGE and anti-HA (αHA) or anti-MBP (αMBP) immunoblotting. The open and closed arrowheads indicate RseP-uncleaved (UC) and -cleaved (CL) forms of each protein. (B) Membrane integration of HA-MBP-RseA140 and its derivatives. Plasmids pSTD797 (RseATM), pSTD800 (LacYTM1) and pSTD843 (LacYTM5) were introduced into KK374 (ΔrseA ΔrseP ΔdegS). Cells were grown as in (A) and converted to spheroplasts, which were treated with 1 mg/ml proteinase K (PK) in the presence or absence of 1% Triton X-100 (TX) as indicated. Proteins were analyzed by SDS–PAGE and anti-HA (αHA), anti-MBP (αMBP) or anti-FtsH (αFtsH) immunoblotting. A cytoplasmic membrane protein FtsH with a large cytoplasmic domain and a protease-resistant periplasmic region was used as an internal control for the spheroplast integrity. The open arrow indicates the intact form of each protein. (C) Pulse-chase assessment of stability of HA-MBP-RseA140 and its derivatives. Cells of AD1811 (rseP⁺) and KK211 (ΔrseP), each carrying pSTD797 (RseATM), pSTD835 (LacYTM1) or pSTD843 (lacYTM5), were grown in M9-glucose (0.4%) medium supplemented with 18 amino acids (other than methionine and cysteine) at 30°C and induced with 1 mM IPTG and 1 mM cAMP for 10 min for the expression of cloned genes. Cells were pulse-labeled with [³⁵S]methionine for 2 min and chased with unlabeled methionine for the indicated periods. Proteins were then precipitated with anti-HA antibodies, separated by SDS–PAGE and visualized by a phosphor imager (BAS1800). (D) RseP-dependent degradation of HA-RseA140 and its derivatives without the MBP moiety. Plasmids pKK58 (encoding HA-RseA140; RseATM), pSTD760 (encoding HA-RseA(LacYTM1)140; LacYTM1) and pSTD767 (encoding HA-RseA(LacYTM5)140; LacYTM5) were introduced into the strains described in (A). Proteins were analyzed as in (A) by anti-HA immunoblotting.

Figure 3

In vitro proteolytic reactions catalyzed by RseP. (A) Purified preparations of RseP-His₆-Myc and the model substrate proteins. A 0.35 μg portion of each of purified samples of RseP-His₆-Myc (lane 1), RseP(H22F)-His₆-Myc (lane 2), His₆-MBP-RseA140 (lane 3), His₆-MBP-RseA(LacYTM1)140 (lane 4), His₆-MBP-RseA(LacYTM1/P28L)140 (lane 5) and His₆-MBP-RseA(A108C)140 (lane 6) was subjected to 10% SDS–PAGE and Coomassie brilliant blue (CBB) staining. Positions of molecular size markers (shown in kDa) are shown on the left. (B) Cleavage of His₆-MBP-RseA140 by RseP. His₆-MBP-RseA140 was incubated with RseP-His₆-Myc (lanes 1–7) or RseP(H22F)-His₆-Myc (lanes 8 and 9) in the presence (lanes 6 and 7) or absence (lanes 1–5, 8 and 9) of 5 mM 1,10-phenanthroline (PT) for the indicated times. 10% SDS–PAGE patterns are shown by CBB staining (upper panel) and anti-MBP immunoblotting (lower panel). (C) Cleavage of His₆-MBP-RseA(LacYTM1)140 and its P28L derivative. RseP-His₆-Myc was incubated with His₆-MBP-RseA(LacYTM1)140 (lanes 1–5) and His₆-MBP-RseA(LacYTM1/P28L)140 (lanes 6 and 7) for the indicated times followed by 10% SDS–PAGE and CBB staining. (D) MalPEG modification of RseP cleavage products of His₆-MBP-RseA140 and His₆-MBP-RseA(A108C)140. RseP-His₆-Myc was incubated with His₆-MBP-RseA140 (lanes 1–4) and His₆-MBP-RseA(A108C)140 (lanes 5–8) for the indicated times. Proteins were then precipitated by trichloroacetic acid treatment, solubilized in 1% SDS and subjected to modification with 5 mM malPEG at 37°C for 1 h. They were analyzed by SDS–PAGE and anti-MBP immunoblotting.

Figure 4

Determination of the RseP cleavage sites within RseATM and LacYTM1. (A) MalPEG modification of engineered cysteines before and after the RseP-dependent cleavage of RseATM variants. Cells expressing an HA-MBP-RseA140 derivative having an engineered cysteine residue in its TM sequence or in the MBP domain were grown in L broth containing 1 mM IPTG and 1 mM cAMP at 30°C. Total cellular proteins were solubilized in 1% SDS, and subjected to modification with 5 mM malPEG at room temperature for 30 min. Subsequently, samples were mixed with an equal volume of 2 × SDS sample buffer and analyzed by SDS–PAGE and anti-HA immunoblotting. The numbering of the amino-acid residues in the RseATM sequence region is according to that of the original RseA protein. (B) MalPEG modification assay to determine the cleavage site of LacYTM1. Cells expressing an HA-MBP-RseA(LacYTM1)140 derivative having an engineered cysteine residue were grown, treated with malPEG and analyzed by SDS–PAGE and anti-HA immunoblotting as in (A). The numbering of the amino-acid residues in the LacYTM1 sequence region is according to that of the original LacY protein. UC and CL indicate the uncleaved and cleaved forms, respectively, of HA-MBP-RseA and its derivatives. The open vertical arrows indicate the site of the RseP cleavage determined from the present results. The LacYTM1 and LacYTM5 sequences with amino-acid residues in a flanking MBP/linker-derived region (shown in italic) are shown on the top of each panel.

Figure 5

RseP-mediated cleavage of proteins having no RseA-related sequence. (A) RseP-dependent cleavage and topology of HA-MBP-LacYTM1-P1^*-Myc. Lanes 1–4: plasmid pSTD853 was introduced into strains AD1811 (ΔrseA, lane 1), KK211 (ΔrseA ΔrseP, lane 2), AD1839 (ΔrseA ΔdegS, lane 3), AD1840 (ΔrseA ΔrseP ΔdegS, lane 4) and KK374 (ΔrseA ΔrseP ΔdegS, lanes 5–7). In vivo cleavage of HA-MBP-LacYTM1-P1^*-Myc by RseP (lanes 1–4) and its topology in the membrane (lanes 5–7) were analyzed by SDS–PAGE and anti-HA immunoblotting as described in the legend to Figure 2. P1^* indicates a region derived from the LacY first periplasmic loop, with amino-acid substitutions introduced into two positions that fortuitously have the same amino-acid residues as the RseA periplasmic residues of the corresponding locations. (B) RseP-mediated cleavage of HA-MBP-SS^Bla-Bla. Total proteins prepared from cells of AD1811 (ΔrseA, lane 1), KK211 (ΔrseA ΔrseP, lane 2), AD1839 (ΔrseA ΔdegS, lane 3) and AD1840 (ΔrseA ΔrseP ΔdegS, lane 4), each carrying pSTD849, were analyzed as above. HA-MBP-SS^Bla indicates the leader peptidase-cleaved product of the full-length protein, whereas its RseP cleavage product is indicated as HA-MBP-SS^Bla*.

Figure 6

Effects of LacYTM1 amino-acid substitutions on the cleavage by RseP. (A) Amino-acid sequences of the TM domains of the HA-MBP-RseA(LacYTM1) derivatives. % cleavage on the right represents the proportion of the RseP-cleaved form in the sum of the cleaved and uncleaved forms of each protein; values obtained in at least two independent experiments are averaged and shown with standard deviations. Residues VD and LQ flanking the LacYTM1 sequence are derived from a SalI and a PstI recognition sequence, respectively. (B) RseP-mediated cleavage and topology of the HA-MBP-RseA(LacYTM1)140 derivatives. RseP-dependent cleavage (lanes 1 and 2) and membrane topology (lanes 3–5) of the HA-MBP-RseA(LacYTM1) derivatives shown in (A) were examined as described in the legend to Figure 2.