Impact of membrane fusion and proteolysis on SpoIIQ dynamics and interaction with SpoIIIAH - PubMed (original) (raw)
Impact of membrane fusion and proteolysis on SpoIIQ dynamics and interaction with SpoIIIAH
Shinobu Chiba et al. J Biol Chem. 2007.
Abstract
The onset of engulfment-dependent gene expression during Bacillus subtilis sporulation requires the forespore membrane protein SpoIIQ, which recruits mother cell proteins involved in late gene expression to the outer forespore membrane. Engulfment activates the late forespore transcription factor sigmaG, which produces high levels of the secreted SpoIVB protease that is required for activation of the late mother cell transcription factor sigmaK. Engulfment also triggers the proteolytic cleavage of SpoIIQ, an event that depends on the SpoIVB protease but not on sigmaG activity. To determine if SpoIVB directly cleaves SpoIIQ and to determine if this event participates in the onset of late gene expression, we purified SpoIVB, SpoIIQ, and SpoIVFA (another SpoIVB substrate). SpoIVB directly cleaved SpoIIQ at the same site in vitro and in vivo and cleaved SpoIVFA in at least three different locations. SpoIIQ cleavage depends on membrane fusion, but not on sigmaG activity, suggesting that the ability of SpoIVB to cleave substrates is regulated by membrane fusion. We isolated SpoIVB-resistant SpoIIQ proteins by random mutagenesis of codons at the cleavage site and demonstrated that SpoIIQ processing is dispensable for spore formation and for activation of late forespore and mother cell gene expression. Fluorescence recovery after photobleaching analysis demonstrated that membrane fusion releases SpoIIQ from an immobile complex, an event that could allow SpoIVB to cleave SpoIIQ. We propose that this membrane fusion-dependent reorganization in the complex, rather than SpoIIQ proteolysis itself, is necessary for the onset of late transcription.
Figures
FIGURE 1. B. subtilis engulfment, SpoIIQ proteolysis, and _σ_G and _σ_K activation
A, after septation, the larger mother cell engulfs the smaller forespore, which is ultimately completely enclosed in the mother cell cytoplasm. GFP-SpoIIQ (dark gray) assembles foci at the septal midpoint and migrates around the forespore with the engulfing membrane, assembling a helical or hexagonal structure in the forespore (19). After engulfment, SpoIIQ is cleaved to release the N-terminal GFP fragment into the forespore cytoplasm (17, 19). The forespore transcription factor _σ_G and the mother cell transcription factor _σ_K are synthesized during engulfment and activated after engulfment (reviewed by Ref. 1). B, the interacting proteins SpoIIQ (Q) and SpoIIIAH (AH) are required for activation of _σ_G and recruit the BofA-SpoIVFA-SpoIVFB complex necessary for activation of _σ_K to the outer forespore membrane (OFM) (17, 24). Activation of _σ_K is governed by two checkpoints: the forespore checkpoint (i) in which _σ_G activation allows the production of high levels of SpoIVB (11), which cleaves SpoIVFA to relieve SpoIVFB from inhibition by BofA, and the engulfment checkpoint (ii) in which _σ_K activity is coupled to engulfment by a BofA-independent mechanism (17). SpoIVB is also required to process SpoIIQ (17). C, SpoIIQ traverses the forespore membrane (FM), with a cytoplasmic N terminus that is the site of the GFP fusion. The extracellular C-terminal domain was used as the antigen for polyclonal antibodies (anti-IIQ) (17) while His6 and FLAG tags were fused to the extreme C terminus. Three proteolysis events are indicated by roman numerals (I–III). The first initiating proteolysis event (I) requires SpoIVB serine protease (lightning bolt), and allows a subsequent cleavage within the membrane or in the cytoplasm (II). There might also be a third cleavage event (III?) near the C terminus of the protein that releases the His6 and FLAG tags. D and E, models for the role of SpoIIQ in engulfment-dependent gene expression. D, the “pre-proteolytic activation model,” in which full-length SpoIIQ is required for _σ_G and _σ_K activity; E, “post-proteolytic activation model,” in which SpoIIQ proteolysis mediates intracellular signal transduction.
FIGURE 2. Identification of SpoIVB cleavage sites in SpoIIQ and SpoIVFA
A, in vivo proteolysis of SpoIIQ (PY79) and SpoIIQ-His6 (SCB9) analyzed by Western blot using anti-SpoIIQ polyclonal antiserum at various times of sporulation. Full-length SpoIIQ (IIQ-FL) migrates at ~37 kDa, the C-terminal product (C-term) at ~26 kDa. B, SpoIIQ-FLAG (lane 2, XJ412) and the negative control strain PY79 (lane 1) were immunoprecipitated with anti-FLAG antibodies, and visualized by Coomassie staining. The bands indicated by C-term in lane 2 and lane 1 were extracted and subject to N-terminal amino acid sequencing. C, in vitro proteolysis of GST-SpoIIQ, various mutant derivatives, and GST-SpoIVFA101–264-FLAG by purified SpoIVB-His6. Purified GST-SpoIIQ43–283 and GST-SpoIVFA were incubated at 37 °C for 4 h in the presence or absence of purified SpoIVB-His6 and visualized by Coomassie staining. IIQ1 and FA1–3 are C-terminal cleavage products of SpoIIQ and SpoIVFA, respectively. G1–3 are fragments containing GST released from the N terminus. D, cleavage sites are indicated by arrowheads. Gray arrowhead 2 indicates the estimated position of the second C-terminal cleavage in SpoIIQ; gray arrowhead 4 indicates cleavage at FA4 (15). E, amino acid alignment of SpoIVB-dependent cleavage sites in SpoIIQ, SpoIVFA, SpoIVB (22), and _α_- and _β_-casein. Arrowhead indicates position of cleavage. The numbers of amino acids before the cleavage site are in parentheses.
FIGURE 3. Protease sensitivity and _σ_G and _σ_K activity of SpoIIQ cleavage site-mutants
A, proteolysis in small-scale cultures. Sporulation was induced by resuspension in a 2-ml culture in test tubes, and samples were prepared after 5 h at 37 °C for Western blot analysis with anti-SpoIIQ. Amino acids introduced at Val-72 (lanes 2–11), Gly-73 (lanes 12–18), and Lys-74 (lanes 19 –27) are indicated. B, time course of proteolysis in large scale cultures. IIQ-FL and C-term indicate full-length SpoIIQ and C-terminal cleavage products. C, alignment of predicted cleavage sites of SpoIIQ from various Bacillus sp. The arrowhead indicates cleavage site of B. subtilis SpoIIQ. D–F, affect of various spoIIQ mutations on _σ_G and σ_K activity. All strains contained the lacZ fusion indicated in each panel, and the spoIIQ mutation indicated by the following symbols (circles, spoIIQ+; squares, Δ_spoIIQ; triangles, wt; inverted triangles, V72C; solid diamonds, V72M; open diamonds, G73V; solid circles, G73E). In addition, all strains with a spoIIQ derivative at amyE also contained a spoIIQ null mutation. D, to assay _σ_G activity, strains KP701, AR232, SCB18, SCB108, SCB109, SCB110, and SCB111 were used. E, for _σ_K activity, XJ220, KP953, SCB21, SCB114, SCB115, SCB116, and SCB117 were used. F, the cleavage site mutants also supported _σ_K activity in strains lacking spoIIIG and bofA. Strains contained the indicated spoIIQ mutations plus bofA and spoIIIG (SCB223, SCB224, SCB225, SCB227, SCB228, and SCB229).
FIGURE 4. Altered proteolysis, localization, and dynamics of cleavage-defective GFP-SpoIIQ
A, proteolysis of GFP-SpoIIQ (SCB6) V72Y (SCB138) and V72E (SCB139), analyzed by Western blots with anti-GFP. “_GFP-Q_” and “_GFP_” indicate full-length and N-terminal GFP degradation product of GFP-SpoIIQ. B, localization of GFP-SpoIIQ (wt) and GFP-SpoIIQ (V72Y) 4 h after the initiation of sporulation (_t_4). Membranes were stained with FM4-64 (red). Arrowheads indicate septal localization of GFP-SpoIIQ (green); double arrows, GFP-SpoIIQ migrating with mother cell membrane; double arrowheads, helical structure. Membrane fusion (indicated by exclusion of FM4-64 from the forespore membranes, arrow and double arrowhead) occurs before proteolysis, which releases GFP into the cytoplasm (arrow). After fusion, V72Y remains membrane-bound but localizes smoothly around the forespore (arrow). C, FRAP analysis of GFP-SpoIIQV72Y at _t_3 performed and quantified as described under “Experimental Procedures.” Images of GFP (green) and FM4-64 (red)-stained membranes of cells before bleaching are to the right of each graph. Images below each plot show the GFP fluorescence during the experiment; the second panel shows the cell just after bleaching. Recovery kinetics were quantified and plotted to show the mean pixel intensity of the bleached (filled square) and unbleached (empty circle) regions and the theoretical pixel intensity value following equilibration between these regions (dashed line). Wild-type GFP-SpoIIQ shows very little recovery (see supplemental Fig. S2 and Ref. 22).
FIGURE 5. The C-terminal fragment of SpoIIQ shows reduced interaction with SpoIIIAH
A and B, sucrose density gradient analysis to assess the apparent molecular masses of proteins in whole cell lysates from _t_2 (A) and _t_3.5 (B). Fractions were collected from the bottom (lane 1) to the top (lane 17) of the gradient. SpoIIIAH-FLAG (AH-flag) and full-length SpoIIQ (IIQ-FL) and the C-terminal cleavage product (C-term) were visualized by Western blot with anti-FLAG or anti-SpoIIQ antibodies, respectively. Arrowheads indicate positions of proteins used as size standards. A, at early times of sporulation (_t_2), full-length SpoIIQ and SpoIIIAH-FLAG are present in the same fractions (~100 kDa). In the absence of one, the other migrates at a lower apparent molecular mass (~40 kDa). B, at later times (_t_3.5), full-length SpoIIQ migrates at ~100 kDa, whereas the C-terminal proteolytic product migrates at a lower apparent molecular mass. C, co-immunoprecipitation of SpoIIQ with SpoIIIAH-FLAG. Whole cell lysates from strains PY79 (spoIIIAH; lanes 1–3) and KP856 (spoIIIAH-flag; lanes 4 – 6) were immunoprecipitated with anti-FLAG M2 antibody. W, B, and U indicate whole cell lysate, bound, and unbound protein fractions, respectively.
FIGURE 6. SpoIIQ proteolysis depends on engulfment membrane fusion
A, SpoIIQ proteolysis assessed by Western blot analysis of strains KP6012 (gfp-spoIIIE+; lanes 1–5) and the membrane fusion-defective KP6111 (_gfp-spoIIIE_121; lanes 6 –10). B and C, schematics depicting the potential importance of coupling SpoIIQ proteolysis to membrane fusion. B, if SpoIIQ proteolysis occurs before fusion, mother cell proteins that interact with SpoIIQ (such as SpoIIIAH) would be released from the septum and therefore be distributed throughout the mother cell membrane. C, if SpoIIQ proteolysis occurs after fusion, binding proteins cannot escape from the outer forespore membrane. Coupling SpoIIQ proteolysis to membrane fusion might therefore be necessary for the efficient localization of mother cell membrane proteins to the outer forespore membrane.
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