Substrate Requirements for Regulated Intramembrane Proteolysis of<i>Bacillus subtilis</i>Pro-σ<sup>K</sup>

Prince, Heather; Zhou, Ruanbao; Kroos, Lee

doi:10.1128/jb.187.3.961-971.2005

Cited by 19 publications

(28 citation statements)

References 73 publications

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“…Previous work showed that deletion of residues 2 to 6 of Pro-K (1-126)-His 6 did not diminish cleavage by SpoIVFB when the two proteins were coexpressed in E. coli (31) (Fig. 3A, lane 1).…”

Section: Resultsmentioning

confidence: 99%

“…3B and C). The protein lacking residues 2 to 20 was included as a control since previous fractionation studies with sporulating B. subtilis suggested that the pro-sequence is required for membrane association of Pro-K (27) or of a fusion protein in which the first 27 residues of Pro-K were fused to the green fluorescent protein (GFP) (31). The marked effect of deleting residues 2 to 20 on the proportion of protein in the IF demonstrates that the assay is sensitive to loss of the pro-sequence.…”

Section: Resultsmentioning

confidence: 99%

“…The sigK null mutant BK410 (47) and its sigK spoIVF null mutant derivative described previously (31) were transformed with plasmids (48) by selection on LB agar containing chloramphenicol (5 g/ml). The plasmids were derived from pDG364, which permits gene replacement of amyE in the chromosome by homologous recombination (double crossover).…”

Section: Plasmid Construction Descriptions Of Plasmid Construction Amentioning

confidence: 99%

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Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

et al. 2013

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bIntramembrane proteases regulate diverse processes by cleaving substrates within a transmembrane segment or near the membrane surface. Bacillus subtilis SpoIVFB is an intramembrane metalloprotease that cleaves Pro-K during sporulation. To elucidate features of Pro-K important for cleavage by SpoIVFB, coexpression of the two proteins in Escherichia coli was used along with cell fractionation. In the absence of SpoIVFB, a portion of the Pro-K was peripherally membrane associated. This portion was not observed in the presence of SpoIVFB, suggesting that it serves as the substrate. Deletion of Pro-K residues 2 to 8, addition of residues at its N terminus, or certain single-residue substitutions near the cleavage site impaired cleavage. Certain multiresidue substitutions near the cleavage site changed the position of cleavage, revealing preferences for a small residue preceding the cleavage site N-terminally (i.e., at the P1 position) and a hydrophobic residue at the second position following the cleavage site C-terminally (i.e., P2=). These features appear to be conserved among Pro-K orthologs. SpoIVFB did not tolerate an aromatic residue at P1 or P2= of Pro-K . A Lys residue at P3= of Pro-K could not be replaced with Ala unless a Lys was provided farther C-terminally (e.g., at P9=). ␣-Helix-destabilizing residues near the cleavage site were not crucial for SpoIVFB to cleave Pro-K . The preferences and tolerances of SpoIVFB are somewhat different from those of other intramembrane metalloproteases, perhaps reflecting differences in the interaction of the substrate with the membrane and the enzyme.

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“…Previous work showed that deletion of residues 2 to 6 of Pro-K (1-126)-His 6 did not diminish cleavage by SpoIVFB when the two proteins were coexpressed in E. coli (31) (Fig. 3A, lane 1).…”

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Plasmid Construction Descriptions Of Plasmid Construction Amentioning

confidence: 99%

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Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

et al. 2013

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“…We found that Eep recognizes the N-terminal (nonpheromone or inhibitor) portion of the signal-sequence-like precursor peptides (amino acids 1 to 16 of the iCF10 precursor sequence) ( Table 3). SpoIVFB requires the N-terminal 117 amino acids of its substrate for RIP-mediated cleavage (42). The region recognized by Eep is much smaller and is more similar to that of the E. coli S2P protein RseP (YaeL), which requires the N-terminal 28 amino acids of its substrate pro-E for RIP-mediated processing (10,31).…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

Chandler

Dunny

2008

J Bacteriol

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Conjugative transfer of the Enterococcus faecalis plasmid pCF10 is induced by the peptide pheromone cCF10 when recipient-produced cCF10 is detected by donors. cCF10 is produced by proteolytic processing of the signal sequence of a chromosomally encoded lipoprotein (CcfA). In donors, endogenously produced cCF10 is carefully controlled to prevent constitutive expression of conjugation functions, an energetically wasteful process, except in vivo, where endogenous cCF10 induces a conjugation-linked virulence factor. Endogenous cCF10 is controlled by two plasmid-encoded products; a membrane protein PrgY reduces pheromone levels in donors, and a secreted inhibitor peptide iCF10 inhibits the residual endogenous pheromone that escapes PrgY control. In this study we genetically determined the amino acid specificity determinants within PrgY, cCF10, and the cCF10 precursor that are necessary for cCF10 processing and for PrgY-mediated control. We showed that amino acid residues 125 to 241 of PrgY are required for specific recognition of cCF10 and that PrgY recognizes determinants within the heptapeptide cCF10 sequence, supporting a direct interaction between PrgY and mature cCF10. In addition, we found that a regulated intramembrane proteolysis (RIP) family pheromone precursor-processing protein Eep recognizes amino acids N-terminal to cCF10 in the signal sequence of CcfA. These results support a model where Eep directly targets pheromone precursors for RIP and PrgY interacts directly with the mature cCF10 peptide during processing. Despite evidence that both PrgY and Eep associate with cCF10 in or near the membrane, results presented here indicate that these two proteins function independently.

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“…K is necessary for association with membranes (28,47,58) and that residues 1-27 fused to GFP allow membrane association upon expression in E. coli (47). Hence, this chimera was designed to test whether the Pro part plus a few residues of K would be sufficient to improve the solubilization of SpoIVFB.…”

Section: Pro-mentioning

confidence: 99%

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

Zhang

Halder

Kerr

et al. 2016

Journal of Biological Chemistry

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Intramembrane metalloproteases (IMMPs) are conserved from bacteria to humans and control many important signaling pathways, but little is known about how IMMPs interact with their substrates. SpoIVFB is an IMMP that cleaves Pro-K during Bacillus subtilis endospore formation. When catalytically inactive SpoIVFB was coexpressed with C-terminally truncated Pro-K (1-126) (which can be cleaved by active SpoIVFB) in Escherichia coli, the substrate dramatically improved solubilization of the enzyme from membranes with mild detergents. Both the Pro(1-20) and K (21-126) parts contributed to improving SpoIVFB solubilization from membranes, but only the K part was needed to form a stable complex with SpoIVFB in a pulldown assay. The last 10 residues of SpoIVFB were required for improved solubilization from membranes by Pro-K (1-126) and for normal interaction with the substrate. The inactive SpoIVFB⅐Pro-K (1-126)-His 6 complex was stable during affinity purification and gel filtration chromatography. Disulfide cross-linking of the purified complex indicated that it resembled the complex formed in vivo. Ion mobility-mass spectrometry analysis resulted in an observed mass consistent with a 4:2 SpoIVFB⅐Pro-K (1-126)-His 6 complex. Stepwise photobleaching of SpoIVFB fused to a fluorescent protein supported the notion that the enzyme is tetrameric during B. subtilis sporulation. The results provide the first evidence that an IMMP acts as a tetramer, give new insights into how SpoIVFB interacts with its substrate, and lay the foundation for further biochemical analysis of the enzyme⅐substrate complex and future structural studies.Many critical cellular processes are regulated by intramembrane proteolysis (1). Intramembrane proteases (IPs) 3 cleave their substrates within a transmembrane segment (TMS) or near the membrane surface. There are three classes of IPs: rhomboids, aspartyl IPs, and IMMPs (often called site-2 proteases or S2Ps). Rhomboids are serine IPs that promote animal cellular signaling, coordinate bacterial quorum sensing, regulate mitochondrial homeostasis, and control protozoan infection (2-5). Presenilin, an aspartyl IP, is the catalytic component of ␥-secretase, which is involved in the processing of the amyloid precursor protein, Notch, and many other subtrates (6, 7). Dysfunction of ␥-secretase contributes to the pathogenesis of Alzheimer disease (8) and many other diseases (7). Aspartyl IPs also include preflagellin and prepilin peptidases involved in bacterial pathogenesis (9), and signal peptide peptidases, which facilitate the clearance of signal peptides from membranes, participate in viral infection, and generate small peptides as signal molecules for immune systems (10, 11). IMMPs also play critical roles in a wide variety of biological functions. In eukaryotes, cholesterol metabolism, the unfolded protein response, and the acute-phase response are regulated by IMMPs (1, 12, 13). In bacteria, IMMPs control sporulation, envelope stress responses, mating signal production, polar morphogenesis, virulence, ...

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Substrate Requirements for Regulated Intramembrane Proteolysis ofBacillus subtilisPro-σ^K

Cited by 19 publications

References 73 publications

Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

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Substrate Requirements for Regulated Intramembrane Proteolysis ofBacillus subtilisPro-σK

Cited by 19 publications

References 73 publications

Features of Pro-σ K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

Features of Pro-σ K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

Characterization of the Sequence Specificity Determinants Required for Processing and Control of Sex Pheromone by the Intramembrane Protease Eep and the Plasmid-Encoded Protein PrgY

Complex Formed between Intramembrane Metalloprotease SpoIVFB and Its Substrate, Pro-σK

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Product

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About

Substrate Requirements for Regulated Intramembrane Proteolysis ofBacillus subtilisPro-σ^K

Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease

Features of Pro-σ ^K Important for Cleavage by SpoIVFB, an Intramembrane Metalloprotease