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• W1998083657 abstract "During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation. During the process of spore formation in Bacillus subtilis, many membrane proteins localize to the polar septum where they participate in morphogenesis and signal transduction. The forespore membrane protein SpoIIQ plays a central role in anchoring several mother-cell membrane proteins in the septal membrane. Here, we report that SpoIIQ is also responsible for anchoring a membrane protein on the forespore side of the sporulation septum. Co-immunoprecipitation experiments reveal that SpoIIQ resides in a complex with the polytopic membrane protein SpoIIE. During the early stages of sporulation, SpoIIE participates in the switch from medial to polar division and co-localizes with FtsZ at the polar septum. We show that after cytokinesis, SpoIIE is released from the septum and transiently localizes to all membranes in the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane on the forespore side. Importantly, the re-localization of SpoIIE to the engulfing septum requires SpoIIQ. These results indicate that SpoIIQ is required to anchor membrane proteins on both sides of the division septum. Moreover, our data suggest that forespore membrane proteins can localize to the septal membrane by diffusion-and-capture as has been described for membrane proteins in the mother cell. Finally, our results raise the intriguing possibility that SpoIIE has an uncharacterized function at a late stage of sporulation. Despite their small size and apparent simplicity, bacteria exhibit remarkably complex spatial organization, in which proteins localize to particular sites within the cell. Well-characterized examples include the cell division proteins, which assemble into a cytokinetic ring at mid-cell (1Barak I. Wilkinson A.J. FEMS Microbiol. Rev. 2007; 31: 311-326Crossref PubMed Scopus (49) Google Scholar, 2Errington J. Daniel R.A. Scheffers D.J. Microbiol. Mol. Biol. Rev. 2003; 67 (table of contents): 52-65Crossref PubMed Scopus (521) Google Scholar, 3Vicente M. Rico A.I. Martinez-Arteaga R. Mingorance J. J. Bacteriol. 2006; 188: 19-27Crossref PubMed Scopus (118) Google Scholar); the chemotaxis receptors, which localize to the flagellated cell pole (4Kentner D. Sourjik V. Curr. Opin. Microbiol. 2006; 9: 619-624Crossref PubMed Scopus (83) Google Scholar); the actin-like cytoskeletal proteins that form spiral-like structures along the long-axis of the cell (5Carballido-Lopez R. Microbiol. Mol. Biol. Rev. 2006; 70: 888-909Crossref PubMed Scopus (154) Google Scholar); and the secretion apparatus, which localizes at discrete sites in the cell membranes (6Campo N. Tjalsma H. Buist G. Stepniak D. Meijer M. Veenhuis M. Westermann M. Muller J.P. Bron S. Kok J. Kuipers O.P. Jongbloed J.D. Mol. Microbiol. 2004; 53: 1583-1599Crossref PubMed Scopus (118) Google Scholar, 7Rosch J. Caparon M. Science. 2004; 304: 1513-1515Crossref PubMed Scopus (118) Google Scholar, 8Shiomi D. Yoshimoto M. Homma M. Kawagishi I. Mol. Microbiol. 2006; 60: 894-906Crossref PubMed Scopus (83) Google Scholar). Although the number of proteins that display specific patterns of localization continues to grow, our knowledge of the cellular landmarks that anchor them at particular sites remains poorly understood. A powerful system in which to identify the anchors responsible for localizing proteins at particular sites is the process of sporulation in Bacillus subtilis. Many proteins involved in spore formation localize to defined subcellular positions and this localization is critical for their activity. Upon the initiation of sporulation, the developing cell (called the sporangium) divides into two compartments of unequal size and dissimilar fates. The smaller compartment (the forespore) matures into a dormant spore, while the larger cell (the mother cell) nurtures the spore, packaging it in a protective coat. Through out the course of this developmental process, the two cells follow completely different programs of gene expression that are set in motion by the compartment-specific transcription factors σF in the forespore and σE in the mother cell. The first landmark event in the process of sporulation is the formation of an asymmetric septum. The switch from medial to polar division is brought about by a change in the subcellular position of the cytokinetic FtsZ-ring from mid-cell to the cell poles (9Levin P.A. Losick R. Genes Dev. 1996; 10: 478-488Crossref PubMed Scopus (220) Google Scholar). The membrane phosphatase SpoIIE, which is synthesized at the onset of sporulation, co-localize with FtsZ and plays an important role in re-positioning the FtsZ ring (10Arigoni F. Pogliano K. Webb C.D. Stragier P. Losick R. Science. 1995; 270: 637-640Crossref PubMed Scopus (147) Google Scholar, 11Barak I. Behari J. Olmedo G. Guzman P. Brown D.P. Castro E. Walker D. Westpheling J. Youngman P. Mol. Microbiol. 1996; 19: 1047-1060Crossref PubMed Scopus (86) Google Scholar, 12Ben-Yehuda S. Losick R. Cell. 2002; 109: 257-266Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 13Khvorova A. Zhang L. Higgins M.L. Piggot P.J. J. Bacteriol. 1998; 180: 1256-1260Crossref PubMed Google Scholar). After the septum is complete, SpoIIE has a second function: the activation of the forespore-specific transcription factor, σF (14Arigoni F. Duncan L. Alper S. Losick R. Stragier P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3238-3242Crossref PubMed Scopus (102) Google Scholar, 15Duncan L. Alper S. Arigoni F. Losick R. Stragier P. Science. 1995; 270: 641-644Crossref PubMed Scopus (197) Google Scholar, 16Feucht A. Magnin T. Yudkin M.D. Errington J. Genes Dev. 1996; 10: 794-803Crossref PubMed Scopus (94) Google Scholar). The localization of SpoIIE at the polar septum plays a critical role in coupling developmental gene expression to the completion of this morphological event (17Rudner D.Z. Losick R. Dev. Cell. 2001; 1: 733-742Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Shortly after polar division, the mother cell engulfs the forespore in a phagocytic-like process generating a cell-within-a-cell. As a result of engulfment, the forespore is surrounded by two membranes: its own referred to as the inner forespore membrane and one derived from the mother cell called the outer forespore membrane. Many proteins involved in spore morphogenesis and signal transduction specifically localize to the engulfing septal membranes (18Abanes-De Mello A. Sun Y.L. Aung S. Pogliano K. Genes Dev. 2002; 16: 3253-3264Crossref PubMed Scopus (95) Google Scholar, 19Blaylock B. Jiang X. Rubio A. Moran Jr., C.P. Pogliano K. Genes Dev. 2004; 18: 2916-2928Crossref PubMed Scopus (84) Google Scholar, 20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar, 21Rubio A. Pogliano K. EMBO J. 2004; 23: 1636-1646Crossref PubMed Scopus (50) Google Scholar, 22Rudner D.Z. Losick R. Genes Dev. 2002; 16: 1007-1018Crossref PubMed Scopus (98) Google Scholar, 23van Ooij C. Losick R. J. Bacteriol. 2003; 185: 1391-1398Crossref PubMed Scopus (50) Google Scholar). Mother cell membrane proteins achieve this localization by insertion into the cytoplasmic membranes followed by diffusion to and capture in the septum (24Rudner D.Z. Pan Q. Losick R.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8701-8706Crossref PubMed Scopus (112) Google Scholar). However, in most cases it is not clear how these proteins are captured at this particular site. One example in which the anchoring mechanism is well understood is the mother-cell membrane protein SpoIIIAH. SpoIIIAH localizes to the engulfing septal membranes and this localization depends on a forespore membrane protein SpoIIQ (19Blaylock B. Jiang X. Rubio A. Moran Jr., C.P. Pogliano K. Genes Dev. 2004; 18: 2916-2928Crossref PubMed Scopus (84) Google Scholar, 20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar). Importantly, the extracellular domains of SpoIIIAH and SpoIIQ interact in the space between the mother cell and forespore membranes (19Blaylock B. Jiang X. Rubio A. Moran Jr., C.P. Pogliano K. Genes Dev. 2004; 18: 2916-2928Crossref PubMed Scopus (84) Google Scholar, 20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar). Thus, the zipper-like interaction between SpoIIQ and SpoIIIAH anchors SpoIIIAH in the engulfing septum. SpoIIQ itself localizes to the septal membrane on the forespore side but the mechanism by which it is anchored at this site is unknown (21Rubio A. Pogliano K. EMBO J. 2004; 23: 1636-1646Crossref PubMed Scopus (50) Google Scholar). Interestingly, SpoIIIAH and SpoIIQ are also required for the proper localization of several other mother-cell membrane proteins (20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar, 25Jiang X. Rubio A. Chiba S. Pogliano K. Mol. Microbiol. 2005; 58: 102-115Crossref PubMed Scopus (36) Google Scholar). However, a direct link between these proteins and SpoIIIAH or SpoIIQ has not been established. In this report, we show that SpoIIQ is responsible for anchoring membrane proteins on the forespore side of the septal membrane. We demonstrate that SpoIIQ resides in a membrane complex with SpoIIE. Re-examination of the subcellular localization of SpoIIE revealed that after asymmetric division, SpoIIE is released from the polar septum and transiently localizes to all membranes of the forespore compartment. Upon the initiation of engulfment, it specifically re-localizes to the septal membrane. We show that SpoIIQ is required to anchor SpoIIE in the engulfing septal membrane. These results indicate that SpoIIQ plays a major role in organizing membrane proteins on both sides of the division septum. Moreover, they suggest that forespore membrane proteins can achieve proper localization by diffusion-and-capture. Finally, these data raise the possibility that SpoIIE has a third function in sporulation during or after the morphological process of engulfment. General Methods–All B. subtilis strains were derived from the prototrophic strain PY79 (26Youngman P.J. Perkins J.B. Losick R. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 2305-2309Crossref PubMed Scopus (202) Google Scholar) and are listed insupplemental Table S1. All strains used for the immunoprecipitation experiments contain a spoIVB mutation to prevent degradation of proteins that have domains in the intermembrane space. 2K. A. Marquis, N. Campo, and D. Z. Rudner, unpublished observations. Sporulation was induced by resuspension at 37 °C according to the method of Sterlini-Mandelstam (27Harwood C.R. Cutting S.M. Molecular Biological Methods for Bacillus. John Wiley & Sons Ltd, Chichester, UK1990Google Scholar). Anti-GFP Antibody Resin–Affinity-purified anti-GFP 3The abbreviations used are: GFPgreen fluorescent proteinPBSphosphate-buffered salineMSmass spectrometryYFPyellow fluorescent proteinCFPcyan fluorescent protein. antibodies (4 mg) (22Rudner D.Z. Losick R. Genes Dev. 2002; 16: 1007-1018Crossref PubMed Scopus (98) Google Scholar) were batched absorbed to 1 ml of protein A-Sepharose (Amersham Biosciences) for 1 h at 4 °C. The antibody resin was washed four times with phosphate-buffered saline (PBS) and the antibody was covalently cross-linked to the protein A-Sepharose by the addition of disuccinimidyl suberate (Pierce) to a final concentration of 5 mm. After 30 min, the reaction was quenched by the addition of Tris, pH 7.5 to a final concentration of 100 mm. The antibody resin was washed with 100 mm glycine pH 2.5 to remove uncross-linked antibody and then neutralized with 1× PBS. green fluorescent protein phosphate-buffered saline mass spectrometry yellow fluorescent protein cyan fluorescent protein. Preparation of Crude Membranes and Detergent Solubilization of Membrane Proteins–50-ml cultures were harvested at hour 2.5 after the initiation of sporulation and washed two times with 1× SMM (0.5 m sucrose, 20 mm MgCl2, 20 mm maleic acid pH 6.5) at room temperature. Cells were resuspended in 1:10 volume 1× SMM and protoplasted with lysozyme (0.5 mg/ml). Protoplasts were collected by centrifugation and flash-frozen in N2(l). Thawed protoplasts were disrupted by osmotic lysis with 3 ml hypotonic buffer (Buffer H) (20 mm Hepes pH 8, 200 mm NaCl, 1 mm dithiothreitol, with protease inhibitors: 1 mm phenylmethylsulfonyl fluoride, 0.5 μg/ml leupeptin, 0.7 μg/ml pepstatin). MgCl2 and CaCl2 were added to 1 mm and lysates were treated with DNAse I (10 μg/ml) (Worthington) and RNAseA (20 μg/ml) (USB) for 1 h on ice. The membrane fraction was separated by centrifugation at 100,000 × g for 1 h at 4 °C. The supernatant was carefully removed, and the membrane pellet was dispersed in 200 μl of Buffer G (Buffer H with 10% glycerol). Crude membranes were aliquoted and flash-frozen in N2(l). 50–100 μl crude membranes were diluted 5-fold with Buffer S (Buffer H with 20% glycerol and 100 μg/ml lysozyme), and membrane proteins were solubilized by the addition of the nonionic detergent digitonin (Sigma) to a final concentration of 0.5%. The mixture was rotated at 4 °C for 1 h. Soluble and insoluble fractions were separated by centrifugation at 100,000 × g for 1 h at 4 °C. Co-immunoprecipitation from Detergent-solubilized Membrane Fractions–The soluble fraction from the digitonin-treated membrane preparation (the load) was mixed with 20 μl of affinity-purified anti-GFP antibody resin and rotated for 4 h at 4 °C. The resin was pelleted at 5 krpm, and the supernatant (the flow-through) was removed. The resin was washed four times with 1 ml of Buffer S + 0.5% digitonin. Immunoprecipitated proteins were eluted by the addition of 50 μl of sodium dodecyl sulfate (SDS) sample buffer (0.25 m Tris, pH 6.8, 6% SDS, 10 mm EDTA, 20% glycerol) and heated for 15 min at 50 °C. The resin was pelleted, and the supernatant (the IP) was transferred to a fresh tube and 2-mercaptoethanol was added to a final concentration of 10%. The load, flow-through, and immunoprecipitate were analyzed by immunoblot and SDS-PAGE followed by silver staining (28Rudner A.D. Hall B.E. Ellenberger T. Moazed D. Mol. Cell. Biol. 2005; 25: 4514-4528Crossref PubMed Scopus (75) Google Scholar). Immunoblot Analysis–Samples were heated for 15 min at 50 °C prior to loading. Proteins were separated by SDS-PAGE on 12% polyacrylamide gels, electroblotted onto polyvinylidene difluoride membranes (PerkinElmer) and blocked in 5% nonfat milk in PBS-0.5% Tween-20. The blocked membrane was probed with anti-GFP (22Rudner D.Z. Losick R. Genes Dev. 2002; 16: 1007-1018Crossref PubMed Scopus (98) Google Scholar), anti-SpoIIQ (29Doan T. Rudner D.Z. Mol. Microbiol. 2007; 64: 500-511Crossref PubMed Scopus (19) Google Scholar), anti-SpoIIE (10Arigoni F. Pogliano K. Webb C.D. Stragier P. Losick R. Science. 1995; 270: 637-640Crossref PubMed Scopus (147) Google Scholar), or anti-EzrA (30Haeusser D.P. Schwartz R.L. Smith A.M. Oates M.E. Levin P.A. Mol. Microbiol. 2004; 52: 801-814Crossref PubMed Scopus (101) Google Scholar) antibodies. Primary antibodies were diluted 1:10,000 into 3% bovine serum albumin in PBS-0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat, anti-rabbit (anti-GFP, anti-SpoIIQ and anti-EzrA) or anti-rat (anti-SpoIIE) immunoglobulin G (Bio-Rad and Pierce) with the Western Lightning substrate (PerkinElmer). Mass Spectrometry Analysis–Individual bands were excised from silver-stained gel and trypsinized. Extracted peptides were then separated on a nanoscale C18 reverse-phase HPLC capillary column, and were subjected to electrospray ionization followed by MS using an LCQ DECA ion-trap mass spectrometer. Fluorescence Microscopy–Fluorescence microscopy was performed with an Olympus BX61 microscope as previously described (31Campo N. Rudner D.Z. J. Bacteriol. 2007; 189: 6021-6027Crossref PubMed Scopus (29) Google Scholar). SpoIIE-GFP (and SpoIIE-YFP) was visualized on agarose pads as described previously (12Ben-Yehuda S. Losick R. Cell. 2002; 109: 257-266Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). All other fluorescent proteins were visualized on glass slides with poly-l-lysine-treated coverslips. Fluorescent signals were visualized with a phase contrast objective UplanF1 100× and captured with a monochrome CoolSnapHQ digital camera (Photometrics) using Metamorph software (Universal Imaging). The lipophylic membrane dye TMA-DPH (Molecular Probes) was used at a final concentration of 0.05 mm and exposure times were typically 200 ms. Images were analyzed, adjusted, and cropped using Metamorph software. SpoIIE Reside in a Membrane Complex with SpoIIQ–The forespore membrane protein SpoIIQ interacts across the sporulation septum with the mother cell membrane protein SpoIIIAH. We and others (19Blaylock B. Jiang X. Rubio A. Moran Jr., C.P. Pogliano K. Genes Dev. 2004; 18: 2916-2928Crossref PubMed Scopus (84) Google Scholar, 20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar) have shown that these two proteins are required for the efficient localization of proteins involved in morphogenesis and cell-cell signaling. To identify other proteins that reside in a complex with SpoIIQ and SpoIIIAH, we immunopurified SpoIIQ from detergent-solubilized membrane fractions. For these experiments, we took advantage of a high affinity anti-GFP antibody resin (32Campo N. Rudner D.Z. Mol. Cell. 2006; 23: 25-35Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) and a strain (BTD665) harboring a functional CFP-SpoIIQ fusion (20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar). At hour 2.5 of sporulation, cells were harvested and disrupted by osmotic lysis. A crude membrane fraction was isolated and solubilized by the addition of the nonionic detergent digitonin. Immunoprecipitations were performed on the clarified detergent-solubilized fraction, and the immunoprecipitates were analyzed by SDS-PAGE followed by silver staining (Fig. 1A and see “Experimental Procedures”). As controls, immunoprecipitations were performed on detergent-solubilized lysates derived from a strain (BNC689) lacking a GFP fusion and a strain (BNC1384) harboring a GFP fusion to an unrelated E. coli membrane protein (MalF) (Fig. 1A) (21Rubio A. Pogliano K. EMBO J. 2004; 23: 1636-1646Crossref PubMed Scopus (50) Google Scholar). The malF-gfp fusion was expressed from the spoIIQ promoter (PspoIIQ). Accordingly, this heterologous membrane protein was synthesized at the same time and in the same cellular compartment as SpoIIQ. Silver staining revealed that several other proteins co-purify with CFP-SpoIIQ (Fig. 1A). Importantly, no more than a few faint bands were detected in the immunoprecipitates from the lysate lacking a GFP fusion (Fig. 1A). Similarly, only a small number of proteins co-immunoprecipitated with the Mal-GFP fusion. We note that the heavy and light chains from the anti-GFP antibodies were barely detectable in these experiments due to the efficient cross-linking procedure we used to generate the antibody resin (see “Experimental Procedures”). To identify the proteins that were present in complexes with SpoIIQ, we cut out individual bands from the silver-stained gel, digested them with trypsin and analyzed the tryptic fragments by mass spectrometry (MS). By this method, we identified several mother-cell-specific proteins and several proteins that are made during vegetative growth. 4N. Campo and D. Z. Rudner, unpublished manuscript. In addition, we identified the sporulation membrane protein SpoIIE. SpoIIE is synthesized at the onset of sporulation prior to asymmetric division and is present in both the mother cell and forespore compartments (33King N. Dreesen O. Stragier P. Pogliano K. Losick R. Genes Dev. 1999; 13: 1156-1167Crossref PubMed Scopus (65) Google Scholar, 34Wu L.J. Feucht A. Errington J. Genes Dev. 1998; 12: 1371-1380Crossref PubMed Scopus (58) Google Scholar, 35York K. Kenney T.J. Satola S. Moran Jr., C.P. Poth H. Youngman P. J. Bacteriol. 1992; 174: 2648-2658Crossref PubMed Scopus (87) Google Scholar). Most of the proteins that co-immunoprecipitated with SpoIIQ also co-purified with CFP-SpoIIIAH in support of the idea that many proteins reside in the zipper complex with SpoIIQ and SpoIIIAH.4 Interestingly, SpoIIE did not co-immunoprecipitate with CFP-SpoIIIAH (supplemental Fig. S1) suggesting that the complex that contains SpoIIQ and SpoIIE is different from the zipper complex that contains SpoIIQ and SpoIIIAH. To validate the MS results, we analyzed the immuno-affinity purifications by immunoblot (Fig. 1B). Anti-GFP antibodies revealed that the purifications were very efficient with greater than 80% of the GFP-fusion proteins immunodepleted from the detergent-solubilized lysates (Fig. 1B, compare load (L) and flow-through (FT)). Immunoblotting with anti-SpoIIE antibodies confirmed that SpoIIE efficiently co-immunoprecipitated with CFP-SpoIIQ but not with MalF-GFP or the untagged control (Fig. 1B). Moreover, an unrelated B. subtilis membrane protein (EzrA) was not detected in any of the immunoprecipitates (Fig. 1B). Finally, to determine whether the conditions used for immunoprecipitation maintained the reported interaction between SpoIIQ and SpoIIIAH (19Blaylock B. Jiang X. Rubio A. Moran Jr., C.P. Pogliano K. Genes Dev. 2004; 18: 2916-2928Crossref PubMed Scopus (84) Google Scholar, 20Doan T. Marquis K.A. Rudner D.Z. Mol. Microbiol. 2005; 55: 1767-1781Crossref PubMed Scopus (99) Google Scholar), we probed the immunopurifications with anti-SpoIIIAH antibodies. Under our conditions, SpoIIIAH remained complexed with CFP-SpoIIQ and was efficiently immunodepleted (supplemental Fig. S2). To more rigorously assess whether SpoIIQ and SpoIIE reside in a complex, we performed a reciprocal immunoprecipitation using a strain (BNC1203) harboring a functional SpoIIE-GFP fusion (33King N. Dreesen O. Stragier P. Pogliano K. Losick R. Genes Dev. 1999; 13: 1156-1167Crossref PubMed Scopus (65) Google Scholar). Analysis of individual bands in the silver-stained gel by MS confirmed the presence of SpoIIQ among the proteins co-purifying with SpoIIE-GFP (Fig. 1A). Immunoblot analysis with anti-SpoIIQ antibodies further supported this conclusion (Fig. 1C). We note that a relatively small amount of SpoIIQ co-immunoprecipitated with SpoIIE-GFP since very little SpoIIQ was immunodepleted from the lysate (Fig. 1C, compare load (L) and flow-through (FT)). Nonetheless, the amount of SpoIIQ present in this immunoprecipitate was significantly higher than in control immunoprecipitations (Fig. 1C). Finally, SpoIIIAH could not be detected by immunoblot in the SpoIIE-GFP immunoprecipitate (data not shown). Altogether, these results indicate that SpoIIE resides in a complex with the forespore membrane protein SpoIIQ. Moreover, our data suggest that the SpoIIQ-SpoIIE complex is different from the zipper complex composed of SpoIIQ and SpoIIIAH. SpoIIE Has a Dynamic Localization Pattern–SpoIIE is a polytopic membrane protein with ten transmembrane segments in the N-terminal portion of the protein and a PP2C-like phosphatase domain in its C terminus (36Adler E. Donella-Deana A. Arigoni F. Pinna L.A. Stragler P. Mol. Microbiol. 1997; 23: 57-62Crossref PubMed Scopus (60) Google Scholar, 37Arigoni F. Guerout-Fleury A.M. Barak I. Stragier P. Mol. Microbiol. 1999; 31: 1407-1415Crossref PubMed Scopus (50) Google Scholar). SpoIIE plays two distinct roles during the early stages of sporulation. First, at the onset of sporulation, SpoIIE participates in the switch from medial to polar septation. Consistent with this activity, SpoIIE-GFP has been shown to co-localize with the cell division protein FtsZ forming polar “E-rings” (10Arigoni F. Pogliano K. Webb C.D. Stragier P. Losick R. Science. 1995; 270: 637-640Crossref PubMed Scopus (147) Google Scholar, 12Ben-Yehuda S. Losick R. Cell. 2002; 109: 257-266Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 33King N. Dreesen O. Stragier P. Pogliano K. Losick R. Genes Dev. 1999; 13: 1156-1167Crossref PubMed Scopus (65) Google Scholar, 38Levin P.A. Losick R. Stragier P. Arigoni F. Mol. Microbiol. 1997; 25: 839-846Crossref PubMed Scopus (68) Google Scholar). Second, after asymmetric division, SpoIIE remains associated with the polar septum (33King N. Dreesen O. Stragier P. Pogliano K. Losick R. Genes Dev. 1999; 13: 1156-1167Crossref PubMed Scopus (65) Google Scholar, 34Wu L.J. Feucht A. Errington J. Genes Dev. 1998; 12: 1371-1380Crossref PubMed Scopus (58) Google Scholar) and plays a critical role in the activation of the forespore-specific transcription factor σF (14Arigoni F. Duncan L. Alper S. Losick R. Stragier P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3238-3242Crossref PubMed Scopus (102) Google Scholar, 15Duncan L. Alper S. Arigoni F. Losick R. Stragier P. Science. 1995; 270: 641-644Crossref PubMed Scopus (197) Google Scholar, 16Feucht A. Magnin T. Yudkin M.D. Errington J. Genes Dev. 1996; 10: 794-803Crossref PubMed Scopus (94) Google Scholar). Because SpoIIQ is synthesized in the forespore under the control of σF and the lysates for our immunoprecipitations were derived from cells at hour 2.5 of sporulation (well after the completion of both events that SpoIIE participates in), it was unexpected to find SpoIIE in a complex with SpoIIQ. With our interaction data in mind, we re-examined the localization pattern of SpoIIE-GFP. As described previously (10Arigoni F. Pogliano K. Webb C.D. Stragier P. Losick R. Science. 1995; 270: 637-640Crossref PubMed Scopus (147) Google Scholar, 33King N. Dreesen O. Stragier P. Pogliano K. Losick R. Genes Dev. 1999; 13: 1156-1167Crossref PubMed Scopus (65) Google Scholar, 34Wu L.J. Feucht A. Errington J. Genes Dev. 1998; 12: 1371-1380Crossref PubMed Scopus (58) Google Scholar), prior to polar septation (hour 1.25), the fusion protein localized in bipolar “E-rings” at both potential division sites (Fig. 2). Surprisingly, immediately after asymmetric division but prior to the initiation of engulfment (flat polar septa), SpoIIE-GFP was not retained at the septum and instead was present throughout the forespore membranes (Fig. 2, hour 1.5). Importantly, this localization pattern was transient and as soon as engulfment initiated (slightly curved polar septa), SpoIIE-GFP re-localized to the septal membranes (Fig. 2, hour 1.75). SpoIIE-GFP remained present in the septal membranes throughout the engulfment process (Fig. 2, hour 2). Finally, when engulfment was nearly complete, weak SpoIIE-GFP foci could be observed in the forespore membranes (Fig. 2, hour 2). The presence of the SpoIIE-GFP fusion so late in sporulation was not due to stabilization of SpoIIE-GFP as has been observed for other GFP fusions (39Resnekov O. Losick R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3162-3167Crossref PubMed Scopus (44) Google Scholar). Native SpoIIE and SpoIIE-GFP were present at similar levels throughout the process of sporulation as assessed by immunoblot (supplemental Fig. S3). Altogether, these results show that the localization of SpoIIE is more dynamic than previously appreciated. We suspect that the transient nature of the intermediate SpoIIE localization pattern likely explains why this dynamic behavior had not been observed previously. Rubio and Pogliano (21Rubio A. Pogliano K. EMBO J. 2004; 23: 1636-1646Crossref PubMed Scopus (50) Google Scholar) have reported previously that all membrane proteins synthesized in the forespore (including heterologous ones like E. coli MalF) localize to the engulfing septal membrane. We therefore wondered whether the septal localization of SpoIIE-GFP during engulfment was specific or part of this default pathway for forespore membrane proteins. To investigate this, we compared the localization of SpoIIE-GFP with one of the membrane proteins (MalF-GFP) used in the original study by Rubio and Pogliano. The localization patterns of these two membrane proteins during sporulation were qualitatively different (supplemental Fig. S4). In the case of SpoIIE-GFP, once engulfment had initiated, virtually all the protein was present in the septal membrane with almost no fluorescence detectable in the cytoplasmic membranes of the forespore (Fig. 2 and supplemental Fig. S4). By contrast, although the" @default.
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