Cytokinesis signals truncation of the PodJ polarity factor by a cell cycle-regulated protease

Abstract

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 PodJ(L) 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 PodJ(L) to a form with altered activity (PodJ(S)). Expression of perP is regulated by a signal transduction system that activates cell type-specific transcription programs and conversion of PodJ(L) to PodJ(S) in response to the completion of cytokinesis. PodJ(S), 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 PodJ(L) substrate cooperatively control the cell cycle-dependent onset of Rip.

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 PodJ_L, whereas green circle indicates PodJ_S. 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 PodJ_L, PodJ_S, 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.

Figure 2

Conversion of PodJ_L to PodJ_S 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 divK_D90G (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 DivK_D90G 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 divK_D90G (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.

Figure 3

Proteolytic processing of PodJ is affected in multiple mutants. (A) Immunoblots show that PodJ_L levels in divK_D90G, ΔdivJ, and ΔpleC mutants differ from that in wild-type cells, regardless of whether podJ is expressed from its native promoter (P_podJ-podJ) or from a xylose-inducible promoter (P_xyl-podJ) on the chromosome. Cells were grown at 30°C. (B) Efficient conversion of PodJ_L to PodJ_S requires perP. Immunoblot on the left compares steady-state levels of PodJ_L and PodJ_S 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 (P_perP-perP). (C) Constitutive perP expression prevents PodJ_L accumulation. perP was placed under the control of a xylose-inducible promoter on the chromosome (P_xyl-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.

Figure 4

Microarray analysis of total RNA in pleC, podJ, divJ, and divK_D90G 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 divK_D90G 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 divK_D90G 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.

Figure 5

Pulse–chase analysis shows inhibition of PodJ_L 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 PodJ_L. Fraction of PodJ_L remaining over time was plotted on a logarithmic scale (base 2) and fitted according to exponential decay. A representative analysis is shown.

Figure 6

PerP cleaves the periplasmic domain of PodJ (PodJ_PERI) in vitro. (A) Schematics depict full-length PerP and PodJ_L and the purified fragments PerP_ΔLS and PodJ_PERI. PerP_ΔLS was tagged with His₆ and T7 epitope at the N-terminus and with His₆ at the C-terminus. PodJ_PERI 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 PerP_ΔLS (lane 1), PodJ_PERI (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 PodJ_PERI by PerP_ΔLS into PodJ_PERI-N and PodJ_PERI-C. The PodJ_PERI-N band is not visible here but is detectable by silver staining (data not shown).

Figure 7

perP transcription is regulated by signaling proteins that affect cell cycle progression. (A) A lacZ gene without its own start codon was translationally fused to the first eight codons of perP (as annotated in GenBank) and transcriptionally fused to a 350-bp upstream sequence. The fusion construct was placed on a low-copy-number vector to generate plasmid pJC327. Partial nucleotide sequence of the perP promoter is shown. Underlining indicates possible CtrA binding site (Ouimet and Marczynski, 2000), whereas box indicates a candidate methylation site (Chen and Shapiro, 2003). Bent arrow indicates the putative transcriptional start site, based on Affymetrix data (PT McGrath et al, unpublished). (B) β-Galactosidase assays indicate variations in perP expression in ΔpleC, divK_D90G, ΔdivJ, and ΔpleC ΔdivJ backgrounds. Mixed populations of cells carrying pJC327 were grown at 30°C and harvested for analysis. β-Galactosidase activities were normalized according to that in wild-type cells. (C) Expression of the lacZ reporter construct was reduced in the ΔpodJ but not the ΔperP strain. (D) perP expression requires active CtrA. Wild-type, ctrA401 (Ts), and cckA1 (Ts) cells carrying pJC327 were grown at the permissive temperature (28°C) and then shifted to the restrictive temperature (37°C). Samples were harvested at 0, 2, and 4 h after the shift to measure β-galactosidase activities, which was normalized according to that in wild-type cells at 0 h. (E) CtrA is a positive regulator of perP transcription. Expression of the lacZ reporter construct was determined in strains carrying an empty vector or a plasmid with ctrA under the control of a xylose-inducible promoter (P_xyl-ctrA). Cells were grown in the presence of glucose and then shifted to media containing xylose. Samples were harvested 0, 2, and 4 h after the shift. Activities were normalized according to that at 0 h in cells carrying the vector alone. Error bars indicate standard deviations.

Figure 8

Model of PodJ_L processing includes a regulatory loop: (1) PodJ_L localizes PleC to the incipient swarmer pole; (2) compartmentalization, monitored in part by PleC, activates PerP expression; and (3) PerP cleaves PodJ_L.