Portal de Periódicos da CAPES

Degradation of MinD oscillator complexes by Escherichia coli ClpXP

2020; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1074/jbc.ra120.013866

1083-351X

Christopher J. LaBreck, Catherine E. Trebino, Colby N. Ferreira, Josiah J. Morrison, Eric C. DiBiasio, Joseph Conti, Jodi L. Camberg,

DNA Repair Mechanisms

MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase–dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation. MinD is a cell division ATPase in Escherichia coli that oscillates from pole to pole and regulates the spatial position of the cell division machinery. Together with MinC and MinE, the Min system restricts assembly of the FtsZ-ring to midcell, oscillating between the opposite ends of the cell and preventing FtsZ-ring misassembly at the poles. Here, we show that the ATP-dependent bacterial proteasome complex ClpXP degrades MinD in reconstituted degradation reactions in vitro and in vivo through direct recognition of the MinD N-terminal region. MinD degradation is enhanced during stationary phase, suggesting that ClpXP regulates levels of MinD in cells that are not actively dividing. ClpXP is a major regulator of growth phase–dependent proteins, and these results suggest that MinD levels are also controlled during stationary phase. In vitro, MinC and MinD are known to coassemble into linear polymers; therefore, we monitored copolymers assembled in vitro after incubation with ClpXP and observed that ClpXP promotes rapid MinCD copolymer destabilization and direct MinD degradation by ClpXP. The N terminus of MinD, including residue Arg 3, which is near the ATP-binding site in sequence, is critical for degradation by ClpXP. Together, these results demonstrate that ClpXP degradation modifies conformational assemblies of MinD in vitro and depresses Min function in vivo during periods of reduced proliferation. Cytokinesis in prokaryotes is a highly organized cellular process wherein a network of widely conserved cell division proteins function together to divide a single bacterial cell into two identical daughter cells (1Egan A.J. Vollmer W. The physiology of bacterial cell division.Ann. NY Acad. Sci. 2013; 1277: 8-28Crossref PubMed Scopus (221) Google Scholar). In Escherichia coli, cell division commences with the assembly of a large ring-like protein structure termed the Z-ring, which contains bundled polymers of the GTPase FtsZ, and FtsZ-interacting proteins including FtsA and ZipA, and serves as the site of constriction (2Lutkenhaus J. Pichoff S. Du S. Bacterial cytokinesis: from Z ring to divisome.Cytoskeleton. 2012; 69: 778-790Crossref PubMed Scopus (194) Google Scholar). The Z-ring is a highly dynamic structure wherein FtsZ subunits are rapidly exchanged with a cytoplasmic pool via cycles of GTP binding and hydrolysis (3Stricker J. Maddox P. Salmon E.D. Erickson H.P. Rapid assembly dynamics of the Escherichia coli FtsZ-ring demonstrated by fluorescence recovery after photobleaching.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3171-3175Crossref PubMed Scopus (291) Google Scholar). Many proteins interact with FtsZ to spatially and temporally regulate Z-ring assembly, and a number of these proteins modulate FtsZ dynamics in the Z-ring (4Viola M.G. LaBreck C.J. Conti J. Camberg J.L. Proteolysis-dependent remodeling of the tubulin homolog FtsZ at the division Septum in Escherichia coli.PLoS One. 2017; 12: e0170505Crossref PubMed Scopus (9) Google Scholar). The Min system of E. coli functions to spatially regulate the site of cell division by inhibiting establishment of the Z-ring near the cell poles. MinD is one of three components of the Min system of proteins in E. coli, which includes MinC, MinD, and MinE. These proteins oscillate across the longitudinal axis of the cell to prevent Z-ring assembly at the poles in E. coli (5Lutkenhaus J. Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring.Ann. Rev. Biochem. 2007; 76: 539-562Crossref PubMed Scopus (418) Google Scholar). The Min system is used by several taxa to regulate division-site selection; however, the oscillation observed in E. coli is not preserved across all organisms that contain a Min system, and some organisms lack a Min system entirely (5Lutkenhaus J. Assembly dynamics of the bacterial MinCDE system and spatial regulation of the Z ring.Ann. Rev. Biochem. 2007; 76: 539-562Crossref PubMed Scopus (418) Google Scholar). MinC directly interacts with FtsZ to disrupt GTP-dependent polymerization in vitro (6Hu Z. Lutkenhaus J. Topological regulation of cell division in Escherichia coli involves rapid pole to pole oscillation of the division inhibitor MinC under the control of MinD and MinE.Mol. Microbiol. 1999; 34: 82-90Crossref PubMed Scopus (354) Google Scholar, 7LaBreck C.J. Conti J. Viola M.G. Camberg J.L. MinC N- and C-domain interactions modulate FtsZ assembly, division site selection, and MinD-dependent oscillation in Escherichia coli.J. Bacteriol. 2019; 201 (e00374-00318)Crossref PubMed Scopus (7) Google Scholar, 8Arumugam S. Petrasek Z. Schwille P. MinCDE exploits the dynamic nature of FtsZ filaments for its spatial regulation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: E1192-E1200Crossref PubMed Scopus (46) Google Scholar). The cellular distribution of MinC is determined by MinD via a direct protein–protein interaction. MinD is a member of the Walker A cytoskeletal ATPases protein family and contains a deviant Walker A motif (9Lowe J. Amos L.A. Evolution of cytomotive filaments: the cytoskeleton from prokaryotes to eukaryotes.Internat. J. Biochem. Cell Biol. 2009; 41: 323-329Crossref PubMed Scopus (101) Google Scholar, 10Michie K.A. Lowe J. Dynamic filaments of the bacterial cytoskeleton.Ann. Rev. Biochem. 2006; 75: 467-492Crossref PubMed Scopus (169) Google Scholar). MinD associates with the cytoplasmic membrane in an ATP-bound dimer conformation via a C-terminal membrane targeting sequence. MinE binds to MinD, stimulating ATP hydrolysis and displacement of MinD from the membrane (11Hu Z. Lutkenhaus J. Topological regulation of cell division in E. coli. spatiotemporal oscillation of MinD requires stimulation of its ATPase by MinE and phospholipid.Mol. Cell. 2001; 7: 1337-1343Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 12Park K.T. Wu W. Battaile K.P. Lovell S. Holyoak T. Lutkenhaus J. The Min oscillator uses MinD-dependent conformational changes in MinE to spatially regulate cytokinesis.Cell. 2011; 146: 396-407Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). MinC and MinD from several organisms, including E. coli, assemble into ATP-dependent cofilaments in vitro (13Ghosal D. Trambaiolo D. Amos L.A. Lowe J. MinCD cell division proteins form alternating copolymeric cytomotive filaments.Nat. Commun. 2014; 5: 5341Crossref PubMed Scopus (49) Google Scholar, 14Conti J. Viola M.G. Camberg J.L. The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide.FEBS Lett. 2015; 589: 201-206Crossref PubMed Scopus (18) Google Scholar, 15Huang H. Wang P. Bian L. Osawa M. Erickson H.P. Chen Y. The cell division protein MinD from Pseudomonas aeruginosa dominates the assembly of the MinC-MinD copolymers.J. Biol. Chem. 2018; 293: 7786-7795Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 16Szewczak-Harris A. Wagstaff J. Lowe J. Cryo-EM structure of the MinCD copolymeric filament from Pseudomonas aeruginosa at 3.1 A resolution.FEBS Lett. 2019; 593: 1915-1926Crossref PubMed Scopus (2) Google Scholar). The Lowe group solved a crystal structure of the Aquifex aeolicus MinCD complex, which supports a model in which A. aeolicus copolymers contain alternating MinC and MinD dimers (13Ghosal D. Trambaiolo D. Amos L.A. Lowe J. MinCD cell division proteins form alternating copolymeric cytomotive filaments.Nat. Commun. 2014; 5: 5341Crossref PubMed Scopus (49) Google Scholar, 14Conti J. Viola M.G. Camberg J.L. The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide.FEBS Lett. 2015; 589: 201-206Crossref PubMed Scopus (18) Google Scholar). In E. coli, residues on the surface of the C-terminal domain of MinC are important for copolymerization with MinD (7LaBreck C.J. Conti J. Viola M.G. Camberg J.L. MinC N- and C-domain interactions modulate FtsZ assembly, division site selection, and MinD-dependent oscillation in Escherichia coli.J. Bacteriol. 2019; 201 (e00374-00318)Crossref PubMed Scopus (7) Google Scholar, 13Ghosal D. Trambaiolo D. Amos L.A. Lowe J. MinCD cell division proteins form alternating copolymeric cytomotive filaments.Nat. Commun. 2014; 5: 5341Crossref PubMed Scopus (49) Google Scholar). Although several groups have reported copolymer formation in vitro, the physiological consequences of MinCD assembly in vivo are largely unknown. The Lutkenhaus group reported that MinD mutants that fail to polymerize with MinC, but still interact with MinC at the membrane, do not result in functional defects in vivo (17Park K.T. Du S. Lutkenhaus J. MinC/MinD copolymers are not required for Min function.Mol. Microbiol. 2015; 98: 895-909Crossref PubMed Scopus (14) Google Scholar). Although copolymers are not essential to complete division in vivo, their assembly may modify Min patterning or oscillation rates in vivo through direct competition of accessible MinD surfaces by MinC and MinE (7LaBreck C.J. Conti J. Viola M.G. Camberg J.L. MinC N- and C-domain interactions modulate FtsZ assembly, division site selection, and MinD-dependent oscillation in Escherichia coli.J. Bacteriol. 2019; 201 (e00374-00318)Crossref PubMed Scopus (7) Google Scholar). In several prokaryotes, cytokinesis is regulated proteolytically by the two-component ATP-dependent protease ClpXP (18Camberg J.L. Wickner S. Regulated proteolysis as a force to control the cell cycle.Structure. 2012; 20: 1128-1130Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar). In E. coli, targeted degradation of FtsZ by ClpXP modulates Z-ring dynamics during the division process (4Viola M.G. LaBreck C.J. Conti J. Camberg J.L. Proteolysis-dependent remodeling of the tubulin homolog FtsZ at the division Septum in Escherichia coli.PLoS One. 2017; 12: e0170505Crossref PubMed Scopus (9) Google Scholar, 19Camberg J.L. Viola M.G. Rea L. Hoskins J.R. Wickner S. Location of dual sites in E. coli FtsZ important for degradation by ClpXP; one at the C-terminus and one in the disordered linker.PLoS One. 2014; 9: e94964Crossref PubMed Scopus (23) Google Scholar, 20Camberg J.L. Hoskins J.R. Wickner S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10614-10619Crossref PubMed Scopus (95) Google Scholar). Additional E. coli cell division proteins have also been identified as ClpXP proteolysis substrates, including ZapC (21Buczek M.S. Cardenas Arevalo A.L. Janakiraman A. ClpXP and ClpAP control the Escherichia coli division protein ZapC by proteolysis.Microbiol. 2016; 162: 909-920Crossref PubMed Scopus (9) Google Scholar) and MinD, which was previously implicated as a substrate in a proteomics study performed under DNA damage conditions (22Neher S.B. Villen J. Oakes E.C. Bakalarski C.E. Sauer R.T. Gygi S.P. Baker T.A. Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.Mol. Cell. 2006; 22: 193-204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.Mol. Cell. 2003; 11: 671-683Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). ClpXP contains both an unfoldase, ClpX, which is a hexameric ring-like AAA+ (ATPase Associated with diverse cellular Activities) ATPase, and the compartmentalized serine protease, ClpP, which is composed of two stacked heptameric rings (24Sauer R.T. Bolon D.N. Burton B.M. Burton R.E. Flynn J.M. Grant R.A. Hersch G.L. Joshi S.A. Kenniston J.A. Levchenko I. Neher S.B. Oakes E.S. Siddiqui S.M. Wah D.A. Baker T.A. Sculpting the proteome with AAA(+) proteases and disassembly machines.Cell. 2004; 119: 9-18Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). The ClpX unfoldase contains an N-terminal substrate-binding domain, also called the zinc-binding domain, that can undergo dimerization in solution and engages some substrates, including FtsZ and phage lambda O (λO) protein, in addition to substrate-specific adaptor proteins, such as SspB (20Camberg J.L. Hoskins J.R. Wickner S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10614-10619Crossref PubMed Scopus (95) Google Scholar, 25Thibault G. Houry W.A. Role of the N-terminal domain of the chaperone ClpX in the recognition and degradation of lambda phage protein O.J. Phys. Chem. B. 2012; 116: 6717-6724Crossref PubMed Scopus (14) Google Scholar, 26Thibault G. Yudin J. Wong P. Tsitrin V. Sprangers R. Zhao R. Houry W.A. Specificity in substrate and cofactor recognition by the N-terminal domain of the chaperone ClpX.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 17724-17729Crossref PubMed Scopus (27) Google Scholar, 27Wojtyra U.A. Thibault G. Tuite A. Houry W.A. The N-terminal zinc binding domain of ClpX is a dimerization domain that modulates the chaperone function.J. Biol. Chem. 2003; 278: 48981-48990Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). After engagement of a substrate, ClpX uses ATP hydrolysis to unfold and translocate substrates through its axial channel into the central chamber of ClpP for degradation (28Grimaud R. Kessel M. Beuron F. Steven A.C. Maurizi M.R. Enzymatic and structural similarities between the Escherichia coli ATP- dependent proteases, ClpXP and ClpAP.J. Biol. Chem. 1998; 273: 12476-12481Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 29Wang J. Hartling J.A. Flanagan J.M. The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis.Cell. 1997; 91: 447-456Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar). Furthermore, ClpXP is a major regulator of protein stability and intracellular protein levels during stationary-phase adaptation and other stress conditions (23Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.Mol. Cell. 2003; 11: 671-683Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 30Weichart D. Querfurth N. Dreger M. Hengge-Aronis R. Global role for ClpP-containing proteases in stationary-phase adaptation of Escherichia coli.J. Bacteriol. 2003; 185: 115-125Crossref PubMed Scopus (87) Google Scholar). Here, to determine if E. coli ClpXP regulates Min system function in E. coli by direct degradation of MinD and to understand how ClpX targets MinD, we reconstituted in vitro degradation assays with MinD and MinD-containing complexes, including MinCD copolymers. We show that ClpXP degrades MinD and destabilizes MinCD copolymers in vitro. Destabilization of MinCD copolymers is another example, in addition to FtsZ, which shows that ClpXP-dependent remodeling and degradation of large polymeric protein assemblies lead to their disassembly. We further demonstrate that ClpXP degrades MinD during stationary phase and that intracellular ClpXP levels modify Min function in vivo. MinD was previously identified as a substrate for ClpXP degradation in E. coli W3110 (22Neher S.B. Villen J. Oakes E.C. Bakalarski C.E. Sauer R.T. Gygi S.P. Baker T.A. Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.Mol. Cell. 2006; 22: 193-204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). In vivo, deletion of either clpX or clpP from a minC deletion strain in E. coli MG1655 leads to a synthetic filamentous phenotype during exponential growth (31Camberg J.L. Hoskins J.R. Wickner S. The interplay of ClpXP with the cell division machinery in Escherichia coli.J. Bacteriol. 2011; 193: 1911-1918Crossref PubMed Scopus (34) Google Scholar). FtsZ, the major component of the Z-ring, is also degraded by ClpXP, and an imbalance of FtsZ levels leads to filamentation (4Viola M.G. LaBreck C.J. Conti J. Camberg J.L. Proteolysis-dependent remodeling of the tubulin homolog FtsZ at the division Septum in Escherichia coli.PLoS One. 2017; 12: e0170505Crossref PubMed Scopus (9) Google Scholar, 20Camberg J.L. Hoskins J.R. Wickner S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10614-10619Crossref PubMed Scopus (95) Google Scholar). To further understand how degradation of MinD by ClpXP impacts Min function during division, we first developed an in vitro degradation assay using purified proteins. MinD (6 μM) was incubated with ClpX (1.0 μM), ClpP (1.2 μM), and ATP for 3 h, and degradation was measured by monitoring the loss of full-length MinD in the reaction with time (Fig. 1A). After incubation of MinD with ClpXP and ATP, we detected 45.9% less MinD after 3 h (Fig. 1A); however, when either ATP or ClpP was omitted from reactions, the level of MinD did not change over the course of the experiment indicating that MinD is degraded by ClpXP in an ATP-dependent manner (Fig. 1A). To quantitatively measure the rate of degradation in reconstituted reactions in vitro, we labeled MinD with Alexa fluor 488 and measured degradation by monitoring fluorescent peptides released following incubation with ClpXP and ATP. Degradation reactions containing ClpXP (0.8 μM), ATP (8 mM), and MinD (10 μM) were stopped by the addition of EDTA (50 mM), and fluorescent peptides were collected by ultrafiltration and quantified by fluorescence. We observed that the amount of MinD degraded increased linearly over the course of 30 min (Fig. 1B). Next, we examined the rate of MinD degradation by ClpXP with increasing MinD concentration (0–16 μM). We observed a concentration-dependent increase in the rate of MinD degradation, which plateaus near 20 μM MinD, with a rate of 0.08 ± 0.01 min−1 (Fig. 1C). The degradation rate of another substrate, FtsZ, has also been shown to increase with increasing substrate concentration, which suggests a low-affinity interaction at low substrate concentrations (4Viola M.G. LaBreck C.J. Conti J. Camberg J.L. Proteolysis-dependent remodeling of the tubulin homolog FtsZ at the division Septum in Escherichia coli.PLoS One. 2017; 12: e0170505Crossref PubMed Scopus (9) Google Scholar, 20Camberg J.L. Hoskins J.R. Wickner S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10614-10619Crossref PubMed Scopus (95) Google Scholar). Together, these results demonstrate that ClpXP recognizes and degrades MinD in a concentration-dependent manner. In the presence of ATP, MinD binds to E. coli phospholipids by inserting a C-terminal amphipathic helix into the phospholipid bilayer and recruits MinC to phospholipids in vitro. MinE also binds to membrane-associated MinD in vitro. MinE stimulates ATP hydrolysis by MinD in the presence of phospholipids, and then MinD dissociates from the phospholipid bilayer (32Hu Z. Gogol E.P. Lutkenhaus J. Dynamic assembly of MinD on phospholipid vesicles regulated by ATP and MinE.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6761-6766Crossref PubMed Scopus (233) Google Scholar, 33Lackner L.L. Raskin D.M. de Boer P.A. ATP-dependent interactions between Escherichia coli Min proteins and the phospholipid membrane in vitro.J. Bacteriol. 2003; 185: 735-749Crossref PubMed Scopus (143) Google Scholar). Because MinD is capable of binding directly to MinC, MinE, and phospholipids, we tested if the addition of E. coli phospholipids, prepared as small unilamellar vesicles (SUVs), modifies the rate of MinD degradation in the absence and the presence of MinC and MinE. We observed that during the interaction with SUVs, MinC (5 μM) or MinE (10 μM) had no significant impact on the rate of MinD degradation by ClpXP under the conditions tested (Fig. 1D). Finally, to confirm that ClpXP does not also degrade MinC or MinE, we monitored degradation of both MinC and MinE, but detected no proteolysis of either protein after 3 h, and autoproteolysis of ClpX was observed (Fig. 1E). MinC and MinD from E. coli, Pseudomonas aeruginosa, and A. aeolicus readily form copolymers in the presence of ATP composed of alternating dimers (13Ghosal D. Trambaiolo D. Amos L.A. Lowe J. MinCD cell division proteins form alternating copolymeric cytomotive filaments.Nat. Commun. 2014; 5: 5341Crossref PubMed Scopus (49) Google Scholar, 14Conti J. Viola M.G. Camberg J.L. The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide.FEBS Lett. 2015; 589: 201-206Crossref PubMed Scopus (18) Google Scholar, 15Huang H. Wang P. Bian L. Osawa M. Erickson H.P. Chen Y. The cell division protein MinD from Pseudomonas aeruginosa dominates the assembly of the MinC-MinD copolymers.J. Biol. Chem. 2018; 293: 7786-7795Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar, 16Szewczak-Harris A. Wagstaff J. Lowe J. Cryo-EM structure of the MinCD copolymeric filament from Pseudomonas aeruginosa at 3.1 A resolution.FEBS Lett. 2019; 593: 1915-1926Crossref PubMed Scopus (2) Google Scholar). ClpXP is known to disassemble FtsZ polymers in vitro (4Viola M.G. LaBreck C.J. Conti J. Camberg J.L. Proteolysis-dependent remodeling of the tubulin homolog FtsZ at the division Septum in Escherichia coli.PLoS One. 2017; 12: e0170505Crossref PubMed Scopus (9) Google Scholar, 20Camberg J.L. Hoskins J.R. Wickner S. ClpXP protease degrades the cytoskeletal protein, FtsZ, and modulates FtsZ polymer dynamics.Proc. Natl. Acad. Sci. U. S. A. 2009; 106: 10614-10619Crossref PubMed Scopus (95) Google Scholar); therefore, we tested if ClpXP could also prevent assembly of alternating MinCD copolymers or destabilize them after they assemble. First, to test if the presence of ClpXP in a MinCD assembly reaction reduces or prevents copolymerization of MinD with ATP and MinC in vitro, we monitored 90° light scatter of reactions containing MinD (8 μM), MinC (4 μM), and then added ATP alone or with ClpX and/or ClpP, where indicated (Fig. 2, A–B). Light scatter was then monitored for an additional 30 min. The addition of ATP without ClpXP stimulated robust copolymer formation; however, the addition of ATP and ClpXP lead to a small increase in light scatter that rapidly decreased with time (Fig. 2A). Next, we tested if ClpX impairs MinCD assembly without ClpP because inhibition of MinCD copolymer assembly could potentially result from MinD unfolding by ClpX, but not degradation. Interestingly, we found that the addition of ClpX resulted in a 45% inhibition of copolymerization (Fig. 2B). To determine if the inhibition required ATP hydrolysis, and therefore also ATP-dependent substrate unfolding, or binding only, we tested if the ClpX ATPase mutant protein, ClpX(E185Q), which hexamerizes and binds substrates but does not unfold them, impairs copolymer formation. Similar to wildtype ClpX, we observed a 45% reduction in the light scatter increase in response to ATP addition, suggesting that the binding, but not unfolding, partially reduces MinCD copolymer abundance (Fig. 2B). Together, these results suggest that ClpX prevents MinCD assembly independently of ClpP and ATP hydrolysis, but that ClpXP is substantially more effective for preventing assembly and/or destabilizing MinCD copolymers (Fig. 2, A–B). Addition of an equivalent volume of buffer or ClpP alone does not inhibit copolymer formation in control experiments (Fig. 2B). Together, our results suggest that MinCD copolymer assembly is prevented by ClpXP in vitro and, to a lesser extent, by ClpX. Next to determine if ClpXP destabilizes preassembled MinCD copolymers, we performed an order of addition experiment. First, copolymers were preassembled with ATP, and then ClpXP (0.5–0.9 μM) was added, and we monitored light scatter for another 30 min to detect MinCD copolymers. After assembly of MinCD polymers, addition of ClpXP to the reaction led to a rapid decrease in light scatter that correlated with increased ClpXP concentration (Fig. 2C). In contrast, the addition of buffer, ClpX, or ClpP failed to destabilize assembled MinCD copolymers (Fig. S1A). Finally, we directly visualized copolymers via negative staining transmission electron microscopy (TEM) and compared MinCD copolymer abundance and morphology to copolymers incubated with ClpXP. Consistent with previous reports, we observed MinCD copolymers with ATP (8 mM) (Fig. 2D). Many copolymers were straight and single stranded; however, we also observed bundles of copolymers. Next, reactions containing ClpXP (0.8 μM) with ATP (8 mM) alone or added to MinCD copolymers, preassembled with ATP, were incubated for 15 min, and reaction products were analyzed by TEM. In the presence of ClpXP, we observed fewer copolymers that were shorter and spread more sparsely across the grid (Fig. 2E), compared with copolymers without ClpXP (Fig. 2D). To confirm that polymers were not observed in reactions containing ClpXP alone, we visualized ClpXP (0.9 μM) assembled with ATP (4 mM). We detected a homogeneous population of ClpXP particles (Fig. 2F) and did not observe any polymeric structures. These results suggest that the ClpXP destabilizes MinCD copolymers, and ClpX is sufficient to prevent assembly of MinCD copolymers through binding and is independent of ATP hydrolysis. In the structural model of MinCD, copolymers are comprised of alternating MinC and MinD dimers (13Ghosal D. Trambaiolo D. Amos L.A. Lowe J. MinCD cell division proteins form alternating copolymeric cytomotive filaments.Nat. Commun. 2014; 5: 5341Crossref PubMed Scopus (49) Google Scholar) (Fig. 3A). Therefore, it is possible that copolymer destabilization by ClpXP could arise from degradation of MinC or a failure of MinC to dimerize and/or interact with MinD. As described, we observed no degradation of MinC by SDS–PAGE after incubation with ClpXP and ATP (Fig. 1E); therefore, copolymer disassembly does not likely occur via MinC engagement. In a control experiment, Gfp–ssrA was rapidly degraded by ClpXP (Fig. S1B). Substrate recognition by ClpX is mediated by the presence of different sequence motifs, or degrons, at the N- or C-terminal regions of protein substrates (23Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.Mol. Cell. 2003; 11: 671-683Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar, 34Baker T.A. Sauer R.T. ClpXP, an ATP-powered unfolding and protein-degradation machine.Biochim. Biophys. Acta. 2012; 1823: 15-28Crossref PubMed Scopus (252) Google Scholar). The N terminus of MinD contains amino acids that bear similarity to the N motif-2 consensus motif (M-b-ϕ-ϕ-ϕ-X5-ϕ) identified for ClpX (22Neher S.B. Villen J. Oakes E.C. Bakalarski C.E. Sauer R.T. Gygi S.P. Baker T.A. Proteomic profiling of ClpXP substrates after DNA damage reveals extensive instability within SOS regulon.Mol. Cell. 2006; 22: 193-204Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 23Flynn J.M. Neher S.B. Kim Y.I. Sauer R.T. Baker T.A. Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals.Mol. Cell. 2003; 11: 671-683Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar) (1MARIIV-X5-G12). In the structural model of MinD, Arg 3 is present near the N terminus, outside the dimer interface, and accessible to the surface (Fig. 3A). To determine if this arginine is important for recognition by ClpX, we mutagenized the residue to glutamate and purified MinD(R3E). In degradation reactions with ClpXP in vitro, we observed that 50% of wildtype MinD was degraded in the first 60 min; however, we detected no MinD(R3E) degradation during the experiment under the conditions tested (Fig. 3B). To confirm that MinD(R3E) is not defective for function, we measured the ability of MinE to stimulate ATP hydrolysis of MinD(R3E) in the presence of SUVs. We observed that the ATP hydrolysis rate of MinD(R3E) (8 μM) was stimulated 10-fold by MinE (16 μM) and SUVs (1 mg ml−1), similar to wildtype MinD, suggesting that the amino acid substitution does not impair MinD function (Fig. 3C). MinD(R3E) is defective for degradation by ClpXP but copolymerizes with MinC (Fig. 3D); therefore, we tested if whether copolymers containing MinD(R3E) and MinC are resistant to destabilization by ClpXP. As expected, we observed that ClpXP failed to destabilize preassembled MinC/MinD(R3E) po