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• W2128722658 abstract "Article5 January 2006free access Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease Joseph C Chen Joseph C Chen Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Alison K Hottes Alison K Hottes Department of Developmental Biology, Stanford University, Stanford, CA, USA Department of Electrical Engineering, Stanford University, Stanford, CA, USA Search for more papers by this author Harley H McAdams Harley H McAdams Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Patrick T McGrath Patrick T McGrath Department of Developmental Biology, Stanford University, Stanford, CA, USA Department of Physics, Stanford University, Stanford, CA, USA Search for more papers by this author Patrick H Viollier Patrick H Viollier Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Joseph C Chen Joseph C Chen Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Alison K Hottes Alison K Hottes Department of Developmental Biology, Stanford University, Stanford, CA, USA Department of Electrical Engineering, Stanford University, Stanford, CA, USA Search for more papers by this author Harley H McAdams Harley H McAdams Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Patrick T McGrath Patrick T McGrath Department of Developmental Biology, Stanford University, Stanford, CA, USA Department of Physics, Stanford University, Stanford, CA, USA Search for more papers by this author Patrick H Viollier Patrick H Viollier Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Lucy Shapiro Corresponding Author Lucy Shapiro Department of Developmental Biology, Stanford University, Stanford, CA, USA Search for more papers by this author Author Information Joseph C Chen1, Alison K Hottes1,2, Harley H McAdams1, Patrick T McGrath1,3, Patrick H Viollier4 and Lucy Shapiro 1 1Department of Developmental Biology, Stanford University, Stanford, CA, USA 2Department of Electrical Engineering, Stanford University, Stanford, CA, USA 3Department of Physics, Stanford University, Stanford, CA, USA 4Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH, USA *Corresponding author. Department of Developmental Biology, Stanford University, Beckman Center B300, Stanford, CA 94305, USA. Tel.: +1 650 725 7678; Fax: +1 650 725 7739; E-mail: [email protected] The EMBO Journal (2006)25:377-386https://doi.org/10.1038/sj.emboj.7600935 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We demonstrate that successive cleavage events involving regulated intramembrane proteolysis (Rip) occur as a function of time during the Caulobacter cell cycle. The proteolytic substrate PodJL is a polar factor that recruits proteins required for polar organelle biogenesis to the correct cell pole at a defined time in the cell cycle. We have identified a periplasmic protease (PerP) that initiates the proteolytic sequence by truncating PodJL to a form with altered activity (PodJS). Expression of perP is regulated by a signal transduction system that activates cell type-specific transcription programs and conversion of PodJL to PodJS in response to the completion of cytokinesis. PodJS, sequestered to the progeny swarmer cell, is subsequently released from the polar membrane by the membrane metalloprotease MmpA for degradation during the swarmer-to-stalked cell transition. This sequence of proteolytic events contributes to the asymmetric localization of PodJ isoforms to the appropriate cell pole. Thus, temporal activation of the PerP protease and spatial restriction of the polar PodJL substrate cooperatively control the cell cycle-dependent onset of Rip. Introduction Proteolysis is integral to the regulatory networks of diverse biological systems (Gottesman, 2003; Jenal and Hengge-Aronis, 2003; Ehrmann and Clausen, 2004). In response to specific cues, proteases can stimulate, modulate, or eliminate the activities of their respective substrates. In the α-proteobacterium Caulobacter crescentus, proteolysis contributes to the defined fluctuations of many regulatory and structural proteins over the course of the cell cycle (Figure 1A and B) (Grunenfelder et al, 2001; Ryan et al, 2004; Skerker and Laub, 2004). The swarmer cell cycle begins with a chemotactic swarmer cell, which has pili and a single flagellum at one pole of the cell. After a period of motility, the swarmer cell differentiates into a sessile stalked cell, replacing these polar organelles with a stalk containing holdfast at its tip for surface adherence. The stalked cell prepares for cell division by replicating its chromosome and assembling a flagellum and the pili secretion apparatus at the pole opposite to the stalk. Asymmetric division then produces a stalked cell and a swarmer cell; the swarmer cell synthesizes pili and starts the swarmer cell cycle again, whereas the stalked cell initiates its cell cycle by immediately beginning another round of chromosome replication and division. Timely proteolysis is critical to the normal progression of these orchestrated processes. For example, degradation of the essential CtrA response regulator during the swarmer-to-stalked transition and in the stalked compartment of the dividing cell helps ensure that chromosome replication initiates at those points in the spatial–temporal matrix (Domian et al, 1997; Ryan et al, 2004). By varying the levels of target proteins, proteases can serve as regulatory switches for different physiological states. Figure 1.PodJ levels and localization vary over the cell cycle between wild-type (left) and ΔperP (right) strains. (A) Schematic diagrams depict PodJ localization during the Caulobacter cell cycle. Red circle indicates PodJL, whereas green circle indicates PodJS. SW, swarmer cell with polar pili (straight lines) and flagellum (wavy line); ST, stalked cell; PD, predivisional cell. (B) Cell extracts from a synchronous population of cells were analyzed for the presence of PodJL, PodJS, McpA, and CtrA by immunoblots. Samples were taken every 20 min, as indicated above the blots. Timing of the cell cycle corresponds to that depicted in panel A. Molecular mass standards, in kDa, are indicated to the left. (C) Cells with yfp-podJ replacing the endogenous podJ allele were examined by differential interference contrast (DIC) and fluorescence microscopy. Localization of YFP-PodJ in individual cells is represented in the schematic panels with orange circles. Download figure Download PowerPoint The interplay of proteolysis and other regulatory mechanisms drives the developmental program integral to the Caulobacter cell cycle (Quardokus and Brun, 2003; Holtzendorff et al, 2004). The polarity determinant PodJ contributes a spatial component to this program (Figure 1A and B) (Wang et al, 1993; Viollier et al, 2002a; Hinz et al, 2003; Lawler et al, 2006). The podJ gene encodes a 974-aa protein with a cytoplasmic N-terminal domain, a single transmembrane segment, and a periplasmic C-terminal region. Transcription of podJ is controlled by the master regulators CtrA, GcrA, and DnaA, such that full-length PodJ (PodJL) is synthesized only in the early predivisional cell (Crymes et al, 1999; Holtzendorff et al, 2004; Hottes et al, 2005). PodJL localizes to the incipient swarmer pole (Figure 1A) and recruits factors required for pili biogenesis, including the PleC histidine kinase/phosphatase and components of the pili assembly machinery (Viollier et al, 2002a; Hinz et al, 2003; Lawler et al, 2006). As the cell divides, the periplasmic domain of PodJL is degraded, giving rise to a truncated form (PodJS) that stays attached to the membrane at the flagellated pole of the progeny swarmer cell (Viollier et al, 2002a; Hinz et al, 2003). PodJS is needed for chemotaxis and holdfast formation, presumably because, by analogy to PodJL, it recruits polar factors required for those processes (Viollier et al, 2002a; Lawler et al, 2006). PodJS is cleared from the cell pole during the swarmer-to-stalked transition, before PodJL is synthesized again and localized to the opposite pole (Viollier et al, 2002a; Hinz et al, 2003; Chen et al, 2005). This proteolytic clearance of PodJS depends on the functionally conserved membrane metalloprotease MmpA (Chen et al, 2005). MmpA and its homologs, in both prokaryotes and eukaryotes, participate in regulated intramembrane proteolysis (Rip), which releases membrane-bound substrates from the membrane and has been adapted as a control mechanism in diverse biological systems (Brown et al, 2000; Urban and Freeman, 2002; Weihofen and Martoglio, 2003). In Caulobacter, Rip by MmpA contributes to the asymmetric distribution of PodJ: loss of MmpA inhibits PodJS degradation and leads to bipolar localization of PodJ isoforms (Chen et al, 2005). Thus, the combination of transcriptional regulation, protein localization, and proteolysis restricts the presence of PodJ isoforms to a specific spatial and temporal pattern during the Caulobacter cell cycle. Changes in PodJ's protein levels and localization during the cell cycle correlate with its functions in polar organelle development. PodJ is required to localize the PleC histidine kinase/phosphatase (Viollier et al, 2002a; Hinz et al, 2003), which, together with the DivJ histidine kinase and the DivK response regulator, constitute the basic components of a signal transduction system that monitors cytokinesis (Figure 2A, left) (Sommer and Newton, 1991; Matroule et al, 2004; McGrath et al, 2004). In the predivisional cell, the DivJ kinase is located at the stalked pole, where it phosphorylates the DivK single-domain response regulator; diffusion of phosphorylated DivK to the swarmer pole results in its dephosphorylation by PleC at that pole (Hecht et al, 1995; Wu et al, 1998; Wheeler and Shapiro, 1999; Jacobs et al, 2001; Lam et al, 2003; Matroule et al, 2004). Hence, DivJ and PleC act in opposition, spatially and catalytically, on DivK's phosphorylation state between the two cell poles (Matroule et al, 2004; McGrath et al, 2004). Cytokinesis, resulting in the formation of a diffusion barrier (Judd et al, 2003), traps dephosphorylated DivK in the swarmer compartment, leading to induction of a swarmer-specific developmental program in that compartment (Matroule et al, 2004). If cell compartmentalization is blocked, dephosphorylated DivK fails to accumulate, and the swarmer-specific program does not initiate (Matroule et al, 2004). PodJ's recruitment of PleC to the correct pole is central to the functionality of this DivJ–PleC–DivK signaling system. Null alleles of podJ and pleC cause similar developmental defects: for example, pili synthesis, a component of swarmer progeny development that occurs after cell division, is abolished in both pleC and podJ null mutants (Wang et al, 1993; Viollier et al, 2002a, 2002b; Hinz et al, 2003). Figure 2.Conversion of PodJL to PodJS depends on compartmentalization, as signaled by DivJ, PleC, and DivK. (A) Schematic diagrams depict the subcellular locations of DivK, PleC, DivJ, and PodJ molecules in wild-type (left) and divKD90G (right) strains. Cytokinesis occurs in the presence of the essential cell division protein FtsZ, but is inhibited in its absence. Wild-type DivK requires compartmentalization for release from the swarmer pole, whereas DivKD90G bypasses that requirement, allowing the swarmer pole to develop a stalk even when division is blocked (Matroule et al, 2004). (B) Immunoblots show contrasting PodJ levels in wild-type (left) and divKD90G (right) cells in the presence (+) or absence (−) of FtsZ. Asynchronous cultures of cells with ftsZ under the control of a chromosomal xylose-regulated promoter were grown in the presence of xylose at 30°C. The cells were then washed and resuspended in media containing glucose or xylose to inhibit or induce FtsZ production, respectively. Samples were taken every hour, starting immediately after the wash. Download figure Download PowerPoint We report here that proteolytic processing of PodJL to PodJS is an integral component of the swarmer development program, initiated by the DivK response regulator after completion of cytokinesis. We also show that the DivJ–PleC–DivK monitoring system controls the expression of a newly identified gene, perP, which encodes a periplasmic protease required for efficient truncation of PodJL. Cell cycle-dependent transcription of perP plays a critical role in regulating its function, as untimely expression of perP prevents PodJL accumulation and, consequently, inhibits pili formation. In addition, perP expression is activated by the CtrA response regulator, reinforcing the hypothesis that CtrA integrates signals from DivJ, PleC, and DivK (Wu et al, 1998; Sciochetti et al, 2002; Ausmees and Jacobs-Wagner, 2003). Because PodJ is required for PleC localization, polar PleC activity affects perP expression, and PerP promotes PodJL processing, we propose a novel regulatory mechanism in which a sequential proteolytic pathway involving Rip is under temporal and spatial control. Results Proteolytic conversion of PodJL to PodJS depends on DivK signaling the completion of cytokinesis Given that proteolytic conversion of PodJL to PodJS occurs at the time of cell division, and that PodJS is the only isoform present in swarmer cells (Viollier et al, 2002a; Hinz et al, 2003), we asked if PodJL processing is a component of the swarmer cell-specific developmental program. First, as determined by Quardokus and Brun (2004), we confirmed that PodJL processing depends on cytoplasmic compartmentalization (Figure 2B, left). Steady-state levels of PodJ isoforms were monitored after inhibition of cytokinesis using a strain in which the essential cell division gene ftsZ is under the control of a xylose-inducible promoter (Wang et al, 2001). In the presence of xylose, cells divided normally, and PodJ levels stayed constant. When xylose was removed, depleting FtsZ and blocking cell division, PodJS levels dropped dramatically, suggesting that PodJL was no longer being converted to PodJS, while any remaining PodJS disappeared because it continued to be degraded. Knowing that the D90G mutation (divK341 allele) in the DivK response regulator can relieve the cytokinesis dependence of swarmer cell development (Figure 2A, right) (Sommer and Newton, 1991; Hecht et al, 1995; Hung and Shapiro, 2002; Matroule et al, 2004), we next examined PodJ levels in the divKD90G mutant when FtsZ was depleted. In the mutant, levels of PodJL and PodJS in non-dividing cells remained comparable to the levels in dividing cells (Figure 2B, right), indicating that PodJL processing bypassed the requirement for compartmentalization. Thus, proteolytic processing of PodJL appears to be a developmental event that occurs in response to DivK signaling the completion of cytokinesis and the creation of separate swarmer and stalked progeny compartments. The distinct phosphorylation states of DivK in the two progeny compartments, generated by the antagonistic effects of the DivJ kinase at the stalked pole and the PleC phosphatase at the swarmer pole, form the basis of the cytokinesis signal (Matroule et al, 2004; McGrath et al, 2004). To confirm that the DivJ–PleC–DivK monitoring system regulates PodJL processing, we compared steady-state levels of PodJL in cells with mutations in divK, divJ, or pleC to that in wild-type cells (Figure 3A, left). In the divKD90G and ΔdivJ mutants, PodJL levels were reduced relative to the wild-type standard, as expected: both mutations tilt the equilibrium toward premature initiation of the swarmer cell-specific program, including PodJL processing. DivKD90G itself does not localize to the flagellar pole, while dephosphorylated DivK predominates in the absence of DivJ and also fails to localize (Wheeler and Shapiro, 1999; Jacobs et al, 2001; Lam et al, 2003; Matroule et al, 2004). These conditions mimic the situation when dephosphorylated DivK is released from the flagellar pole in the swarmer compartment following cell division (Jacobs et al, 2001; Matroule et al, 2004). Figure 3.Proteolytic processing of PodJ is affected in multiple mutants. (A) Immunoblots show that PodJL levels in divKD90G, ΔdivJ, and ΔpleC mutants differ from that in wild-type cells, regardless of whether podJ is expressed from its native promoter (PpodJ-podJ) or from a xylose-inducible promoter (Pxyl-podJ) on the chromosome. Cells were grown at 30°C. (B) Efficient conversion of PodJL to PodJS requires perP. Immunoblot on the left compares steady-state levels of PodJL and PodJS in wild-type, ΔperP, ΔmmpA, and ΔmmpA ΔperP strains. Immunoblot on the right shows PodJ levels in wild-type and ΔperP strains when they carry a complementing plasmid (+) or the vector alone (−). The complementing plasmid contains perP under the control of its own promoter (PperP-perP). (C) Constitutive perP expression prevents PodJL accumulation. perP was placed under the control of a xylose-inducible promoter on the chromosome (Pxyl-perP). Wild-type, ΔperP, ΔmmpA, and ΔmmpA ΔperP strains carrying the construct were grown in the presence (+) or absence (−) of xylose and harvested for immunoblot analysis. (D) Schematic diagram depicts sequential proteolytic processing of PodJ, first by PerP, then by MmpA, and finally by an unknown cytoplasmic protease. Download figure Download PowerPoint On the other hand, the ΔpleC mutation leads to accumulation of phosphorylated DivK, preventing its release from the flagellated pole and initiation of swarmer cell development (Wheeler and Shapiro, 1999; Jacobs et al, 2001; Matroule et al, 2004). As a consequence, PodJL processing should be inhibited. Indeed, we observed accumulation of PodJL in ΔpleC cells (Figure 3A). This elevated level of PodJL was reduced when the ΔdivJ mutation was combined with the ΔpleC mutation (Figure 3A), because DivJ and PleC act in opposition on DivK. We also compared PodJS levels in the various mutants to that in wild-type and ΔmmpA cells (Figure 3A). As previously reported (Chen et al, 2005), PodJS degradation was inhibited in the ΔmmpA mutant, leading to higher steady-state levels, but it did not differ significantly from wild type in strains with mutations in divK, divJ, or pleC. To eliminate the possibility that variation in PodJL levels resulted from changes in podJ transcription, we placed podJ under the control of a xylose promoter on the chromosome and examined protein levels when the gene was constitutively induced (Figure 3A, right). Similar results were obtained as when podJ was under the control of its own promoter. Therefore, the DivJ–PleC–DivK signaling pathway does not modulate PodJL levels by affecting podJ expression; rather, it may regulate post-transcriptional events such as proteolytic processing of PodJL. A periplasmic protease participates in PodJL processing We postulated that the DivJ–PleC–DivK system controls the expression of factors that regulate PodJL processing. To find such factors, we performed microarray analysis on pleC, podJ, and divJ mutants, comparing their steady-state mRNA levels to that in wild-type cells (Figure 4). We identified a set of 26 genes whose transcript levels appeared reduced in pleC and podJ and elevated in divJ mutants. In addition, the expression patterns of these genes during the Caulobacter cell cycle were consistent with participation in the swarmer progeny program (Figure 4) (Laub et al, 2000): their mRNA levels were elevated in swarmer cells, dropped during the swarmer-to-stalked transition, and stayed low until the time of cell division. The expression patterns of these swarmer-specific genes were also similar over the course of a modified cell cycle in the divKD90G mutant (Figure 4). The divKD90G mutant was isolated as a cold-sensitive extragenic suppressor of a pleC mutation (Sommer and Newton, 1991) and, at the restrictive temperature, fails to degrade the master response regulator CtrA and to initiate chromosome replication (Hung and Shapiro, 2002). When divKD90G swarmer cells were allowed to start the cell cycle synchronously at the restrictive temperature, transcript levels of these swarmer-specific genes stayed elevated, as the cell cycle was stalled due to the continual presence of active CtrA. Only when the cells were shifted to the permissive temperature, allowing CtrA to be degraded and the cell cycle to progress, did the transcript levels drop. Thus, the DivJ–PleC–DivK pathway appears to trigger expression of this set of genes in the swarmer compartment. Figure 4.Microarray analysis of total RNA in pleC, podJ, divJ, and divKD90G mutants shows variations in expression profiles. For the pleC, podJ, and divJ strains, RNA was extracted from asynchronous populations of mutant cells and compared to that of wild-type CB15. Blue indicates a decrease and yellow indicates an increase relative to the CB15 reference. For the divKD90G mutant, swarmer cells were isolated and grown at the restrictive temperature (18°C) for 300 min, then shifted to the permissive temperature (33°C) for another 80 min. Samples were taken at the indicated times and compared to reference RNA from a mixed population of wild-type cells grown at 30°C. Expression profiles from the wild-type cell cycle are included for comparison (Laub et al, 2000; Hottes et al, 2005). For the wild-type and divKD90G cell cycles, yellow and blue indicate increase and decrease, respectively, relative to the average expression value for that gene during the cycle. Schematics depict stages of the cell cycle; circle or theta structure in the cell represents quiescent or replicating chromosome, respectively. HK, histidine kinase; RR, response regulator. Download figure Download PowerPoint One member in particular among the set of DivK-regulated genes was a candidate for controlling PodJL processing. The CC1307 gene encodes a protein with a putative signal sequence or membrane anchor at its N-terminus (Bendtsen et al, 2004) and a conserved aspartic protease motif in its periplasmic domain (Marchler-Bauer and Bryant, 2004). As PodJL processing involves truncation of its periplasmic domain (see Figure 3D), we suspected that the CC1307 gene product is the perpetrating protease and named it perP, for periplasmic protease of PodJ. To confirm this, we constructed a strain with an in-frame deletion of perP and examined its PodJ levels by immunoblot analysis (Figure 3B). Compared to that in wild-type cells, PodJL levels were elevated in the ΔperP mutant, whereas PodJS levels were reduced to being almost undetectable, suggesting that conversion of PodJL to PodJS was inhibited in the ΔperP mutant. We were able to complement this PodJL processing defect in the ΔperP mutant by introducing a plasmid carrying perP under the control of its own promoter (Figure 3B, right). However, the complementing plasmid reduced steady-state levels of PodJL in both wild-type and ΔperP strains, raising the possibility that improperly regulated expression of perP from the plasmid affects PodJL accumulation (see below). We also examined PodJ levels in a ΔmmpA ΔperP double mutant and found increased levels of PodJL, similar to that in the ΔperP mutant (Figure 3B). Notably, PodJS levels in the double mutant were similar to that in wild type and intermediate between those in the ΔmmpA and ΔperP single mutants. We interpret these results as follows: PodJL is normally truncated by PerP to produce PodJS, which is then cleaved by MmpA and released from the cell membrane for subsequent degradation in the cytoplasm (Figure 3D) (Chen et al, 2005). Sequential degradation of PodJ by the PerP and MmpA proteases forms the first two steps of the Rip pathway in Caulobacter. In the absence of PerP, conversion of PodJL to PodJS is inhibited, preventing PodJS accumulation. Nevertheless, PodJL may still be processed by proteases other than PerP, albeit significantly less efficiently, and this leaky processing may lead to some PodJS accumulation when PodJS degradation fails to occur in the absence of the MmpA protease. To demonstrate that PerP participates in the proteolytic processing of PodJL, we performed pulse–chase analysis and compared PodJL stability in wild-type and ΔperP strains (Figure 5). In wild-type cells, newly synthesized PodJL stayed relatively stable for about 45 min and then rapidly disappeared, with a half-life of approximately 15 min. As previously reported (Hinz et al, 2003; Chen et al, 2005), the disappearance of PodJL corresponded to the concomitant appearance of PodJS, implicating proteolytic conversion. This stability profile is consistent with PodJL being synthesized in the early predivisional cell and remaining intact until cytoplasmic compartmentalization (Judd et al, 2003), when it is truncated. In the ΔperP mutant, PodJL was relatively stable for at least 120 min after the chase, with negligible appearance of PodJS. These results indicate that PerP is required for the efficient proteolytic conversion of PodJL to PodJS. Figure 5.Pulse–chase analysis shows inhibition of PodJL processing in the ΔperP mutant. Mixed populations of wild-type or ΔperP cells were pulse-labeled with [35S]methionine for 7.5 min and then chased with excess unlabeled methionine and casamino acids. Samples were taken at 15-min intervals after the chase and immunoprecipitated with antibodies to the N-terminal domain of PodJ. Immunoprecipitates were resolved by SDS–PAGE, as shown, to determine the relative levels of PodJL. Fraction of PodJL remaining over time was plotted on a logarithmic scale (base 2) and fitted according to exponential decay. A representative analysis is shown. Download figure Download PowerPoint We determined that PerP directly truncates PodJL by performing in vitro cleavage assays with purified domains of PerP and PodJL (Figure 6). The periplasmic domain of PodJL (PodJPERI) was tagged with His6 at the N-terminus and purified as described previously (Viollier et al, 2002a). PerP, without its first 28 amino acids, including a hydrophobic leader sequence, was tagged with His6 at both termini to generate PerPΔLS. PerPΔLS was purified and cleavage reactions were carried out as described in Materials and methods. In the presence of PerPΔLS, the 33-kDa PodJPERI was cut into two fragments, PodJPERI-C (∼27 kDa) and PodJPERI-N (∼6 kDa); PodJPERI-N was not detectable by Coomassie blue staining but was visible by silver staining (data not shown). Cleavage of PodJPERI appeared to progress linearly with time, and while the assays shown in Figure 6 required long incubation times, there is precedent: in vitro cleavage of anti-sigma factor RseA by the DegS periplasmic protease required 4 h (Walsh et al, 2003). Furthermore, PerPΔLS was purified from inclusion bodies and may have a low fraction of active proteases. We assigned the 27-kDa fragment as the one containing the C-terminus of PodJPERI for two reasons. First, previous studies suggested that removal of 20–30 kDa from PodJL's C-terminus yields PodJS (Viollier et al, 2002a; Chen et al, 2005), and the size of the removed fragment is consistent with that of PodJPERI-C. Second, we examined Escherichia coli cells expressing a PodJL construct containing GFP at its N-terminus by immunoblotting (Supplementary Figure S6). The construct was cut into two fragments when the cells coexpressed full-length PerP: one fragment was only detectable with antibodies to GFP, and the other, exactly the same size as PodJPERI-C, was only detectable with antibodies to the C-terminal domain of PodJL. The 6-kDa fragment (PodJPERI-N) released in the in vitro assay, we believe, remains attached to the transmembrane domain in vivo. Our experiments thus suggest that PerP cleaves PodJL near the N-terminus of its periplasmic domain to produce membrane-bound PodJS and PodJPERI-C (Figure 3D). PodJPERI-C is then presumably cleared from the periplasm by other proteases in vivo. Figure 6.PerP cleaves the periplasmic domain of PodJ (PodJPERI) in vitro. (A) Schematics depict full-length PerP and PodJL and the purified fragments PerPΔLS and PodJPERI. PerPΔLS was tagged with His6 and T7 epitope at the N-terminus and with His6 at the C-terminus. PodJPERI was tagged with His6 at the N-terminus. Numbered arrows point to relevant amino-acid residues. LS, hydrophobic leader sequence; TM, transmembrane segment. (B) Reaction mixtures containing purified PerPΔLS (lane 1), PodJPERI (lane 2), or both (lanes 3–5) were incubated at 37°C for 4 (lane 3), 8 (lane 4), or 20 h (lanes 1, 2, 5). Samples were resolved by SDS–PAGE and visualized by Coomassie blue staining. The MW lane contains molecular weight markers, indicated on the left in kDa. Diagram below the gel shows cleavage of PodJPERI by PerPΔLS into PodJPERI-N and PodJPERI-C. The PodJPERI-N band is not visible here but is detectable by silver staining (data not shown). Download figure Download PowerPoint PerP expression affects PodJL distribution and function To ascertain that PerP is responsible for PodJL processing at a specific time during the Caulobacter cell cycle, we monitored PodJ l" @default.
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• W2128722658 title "Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease" @default.
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• W2128722658 doi "https://doi.org/10.1038/sj.emboj.7600935" @default.
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