The switch I and II regions of MinD are required for binding and activating MinC

MinD and MinC cooperate to form an efficient inhibitor of Z-ring formation that is spatially regulated by MinE. MinD activates MinC by recruiting it to the membrane and targeting it to a septal component. To better understand this activation, we have isolated loss-of-function mutations in minD and carried out site-directed mutagenesis. Many of these mutations block MinC-MinD interaction; however, they also prevent MinD self-interaction and membrane binding, suggesting that they affect nucleotide interaction or protein folding. Two mutations in the switch I region (MinD box) and one mutation in the switch II region had little affect on most MinD functions, such as MinD self-interaction, membrane binding, and MinE stimulation; however, they did eliminate MinD-MinC interaction. Two additional mutations in the switch II region did not affect MinC binding. Further study revealed that one of these allowed the MinCD complex to target to the septum but was still deficient in blocking division. These results indicate that the switch I and II regions of MinD are required for interaction with MinC but not MinE and that the switch II region has a role in activating MinC.     

The MinD membrane targeting sequence is a transplantable lipid-binding helix

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably also Archaea. It was recently demonstrated that membrane localization of MinD is mediated by an 8-12-residue C-terminal motif termed the membrane targeting sequence or MTS. In this study we show that the MinD MTS is a transplantable lipid-binding motif that can effectively target heterologous proteins to the cell membrane. We demonstrate that eubacterial MTSs interact directly with lipid bilayers as an amphipathic helix, with a distinct preference for anionic phospholipids. Moreover, we provide evidence that the phospholipid preference of each MTS, as well as its affinity for biological membranes, has been evolutionarily "tuned" to its specific role in different bacteria. We propose a model to describe how the MTS is coupled to ATP binding to regulate the reversible membrane association of Escherichia coli MinD during its pole-to-pole oscillation cycle.     

Analysis of MinD mutations reveals residues required for MinE stimulation of the MinD ATPase and residues required for MinC interaction

The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.     

A conserved motif within the flexible C-terminus of the translational regulator 4E-BP is required for tight binding to the mRNA cap-binding protein eIF4E

Although the central α-helical Y(X)4LΦ motif (X, variable amino acid; Φ, hydrophobic amino acid) of the translational regulator 4E-BP [eIF (eukaryotic initiation factor) 4E-binding protein] is the core binding region for the mRNA cap-binding protein eIF4E, the functions of its N- and C-terminal flexible regions for interaction with eIF4E remain to be elucidated. To identify the role for the C-terminal region in such an interaction, the binding features of full-length and sequential C-terminal deletion mutants of 4E-BPn (n=1-3) subtypes were investigated by SPR (surface plasmon resonance) analysis and ITC (isothermal titration calorimetry). Consequently, the conserved PGVTS/T motif within the C-terminal region was shown to act as the second binding region and to play an important role in the tight binding to eIF4E. The 4E-BP subtypes increased the association constant with eIF4E by approximately 1000-fold in the presence of this conserved region compared with that in the absence of this region. The sequential deletion of this conserved region in 4E-BP1 showed that deletion of Val81 leads to a considerable decrease in the binding ability of 4E-BP. Molecular dynamics simulation suggested that the conserved PGVTS/T region functions as a kind of paste, adhering the root of both the eIF4E N-terminal and 4E-BP C-terminal flexible regions through a hydrophobic interaction, where valine is located at the crossing position of both flexible regions. It is concluded that the conserved PGVTS/T motif within the flexible C-terminus of 4E-BP plays an auxiliary, but indispensable, role in strengthening the binding of eIF4E to the core Y(X)4LΦ motif.     

Cell division inhibitors SulA and MinC/MinD block septum formation at different steps in the assembly of the Escherichia coli division machinery

SulA and MinCD are specific inhibitors of cell division in Escherichia coli. In this paper, size exclusion chromatography was used to study the effect of the SulA and MinCD division inhibitors on the oligomerization state of endogenous FtsZ in cytoplasmic extracts, and immunofluorescence microscopy was used to determine the effect of SulA and MinCD on the formation of FtsZ, FtsA and ZipA rings at potential division sites. SulA prevented the formation of high-molecular-weight FtsZ polymers by interfering with FtsZ dimerization and subsequent oligomerization. In contrast, the MinCD division inhibitor did not prevent the oligomerization of FtsZ in the cell extracts or the formation of FtsZ and ZipA ring structures in vivo. However, MinCD did prevent the formation of FtsA rings. Increased expression of ftsA suppressed MinCD-induced division inhibition, but had no effect on SulA-induced division inhibition. These results indicate that MinCD blocks the assembly of the septation machinery at a later step than SulA, at the stage at which FtsA is added to the FtsZ ring.     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.     

A domain at the C-terminus of PS1 is required for presenilinase and gamma-secretase activities

The structural requirements for presenilin (PS) to produce active presenilinase and gamma-secretase enzymes are poorly understood. Here we investigate the role the cytoplasmic C-terminal region of PS1 plays in PS1 activity. Deletion or addition of residues at the PS C-terminus has been reported to inhibit presenilinase endoproteolysis of PS and alter gamma-secretase activity. In this study, we use a sensitive assay in PS1/2KO MEFs to define a domain at the extreme C-terminus of PS1 that is essential for both presenilinase and gamma-secretase activities. Progressive deletion of the C-terminus demonstrated that removal of nine residues produces a PS1 molecule (458ST) that lacks both presenilinase processing and gamma-secretase cleavage of Notch and APP substrates. In contrast, removal of four or five residues had no effect (462ST, 463ST), while intermediate truncations partially inhibited PS1 activity. The 458ST mutant was unable to replace endogenous wtPS1 in HEK293 cells. Although 458ST was able to form a gamma-secretase complex, this complex was not matured, illustrated by mutant PS1 instability, lack of endoproteolysis, and little production of mature Nicastrin. These data indicate that the C-terminal end of PS1 is essential for Nicastrin trafficking and modification as well as the replacement of endogenous PS1 by PS1 transgenes.     

The C-terminus of glutathione S-transferase A1-1 is required for entropically-driven ligand binding

Binding of a hydrophobic glutathione product conjugate to rGST A1-1 proceeds via a two-step mechanism, including rapid ligand docking, followed by a slow isomerization to the final [GST.ligand] complex, which involves the localization of the flexible C-terminal helix. These kinetically resolved steps have been observed previously by stopped-flow fluorescence with the wild-type rGST A1-1, which contains a native Trp-21 approximately 20 A from the ligand binding site at the intrasubunit domain-domain interface. To confirm this binding mechanism, as well as elucidate the effects of truncation of the C-terminus, we have further characterized the binding and dissociation of the glutathione-ethacrynic acid product conjugate (GS-EA) to wild-type, F222W:W21F, and Delta209-222 rGST A1-1 and wild-type hGST A1-1. Although modest kinetic differences were observed between the hGST A1-1 and rGST A1-1, stopped-flow binding studies with GS-EA verified that the two-step mechanism of ligand binding is not unique to the GST A1-1 isoform from rat. An F222W:W21F rGST A1-1 double mutant provides a direct fluorescence probe of changes in the environment of the C-terminal residue. The observation of two relaxation times during ligand binding and dissociation to F222W:W21F suggests that the C-terminus has an intermediate conformation following ligand docking, which is distinct from its conformation in the apoenzyme or localized helical state. For the wild-type, Delta209-222, and F222W:W21F proteins, variable-temperature stopped-flow experiments were performed and activation parameters calculated for the individual steps of the binding reaction. Activation parameters for the binding reaction coordinate illustrate that the C-terminus provides a significant entropic contribution to ligand binding, which is completely realized within the initial docking step of the binding mechanism. In contrast, the slow isomerization step is enthalpically driven. The partitioning of entropic and enthalpic components of binding energy was confirmed by isothermal titration calorimetry with wild-type and Delta209-222 rGST A1-1.     

Ergosterol is required for targeting of tryptophan permease to the yeast plasma membrane

It was known that the uptake of tryptophan is reduced in the yeast erg6 mutant, which is defective in a late step of ergosterol biosynthesis. Here, we show that this is because the high affinity tryptophan permease Tat2p is not targeted to the plasma membrane. In wild-type cells, the plasma membrane localization of Tat2p is regulated by the external tryptophan concentration. Tat2p is transported from the Golgi apparatus to the vacuole at high tryptophan, and to the plasma membrane at low tryptophan. However, in the erg6 mutant, Tat2p is missorted to the vacuole at low tryptophan. The plasma membrane targeting of Tat2p is dependent on detergent-insoluble membrane domains, suggesting that sterol affects the sorting through the organization of lipid rafts. The erg6 mutation also caused missorting to the multivesicular body pathway in late endosomes. Thus, sterol composition is crucial for protein sorting late in the secretory pathway. Tat2p is subject to polyubiquitination, which acts as a vacuolar-targeting signal, and the inhibition of this process suppresses the Tat2p sorting defects of the erg6 mutant. The sorting mechanisms of Tat2p that depend on both sterol and ubiquitin will be discussed.     

Determination of the structure of the MinD-ATP complex reveals the orientation of MinD on the membrane and the relative location of the binding sites for MinE and MinC

The three Min proteins spatially regulate Z ring positioning in Escherichia coli and are dynamically associated with the membrane. MinD binds to vesicles in the presence of ATP and can recruit MinC or MinE. Biochemical and genetic evidence indicate the binding sites for these two proteins on MinD overlap. Here we solved the structure of a hydrolytic-deficient mutant of MinD truncated for the C-terminal amphipathic helix involved in binding to the membrane. The structure solved in the presence of ATP is a dimer and reveals the face of MinD abutting the membrane. Using a combination of random and extensive site-directed mutagenesis additional residues important for MinE and MinC binding were identified. The location of these residues on the MinD structure confirms that the binding sites overlap and reveals that the binding sites are at the dimer interface and exposed to the cytosol. The location of the binding sites at the dimer interface offers a simple explanation for the ATP dependence of MinC and MinE binding to MinD.     

Recruitment of MinC,an inhibitor of Z-ring formation,to the membrane in Escherichia coli: role of MinD and MinE

In Escherichia coli, the min system prevents division away from midcell through topological regulation of MinC, an inhibitor of Z-ring formation. The topological regulation involves oscillation of MinC between the poles of the cell under the direction of the MinDE oscillator. Since the mechanism of MinC involvement in the oscillation is unknown, we investigated the interaction of MinC with the other Min proteins. We observed that MinD dimerized in the presence of ATP and interacted with MinC. In the presence of a phospholipid bilayer, MinD bound to the bilayer and recruited MinC in an ATP-dependent manner. Addition of MinE to the MinCD-bilayer complex resulted in release of both MinC and MinD. The release of MinC did not require ATP hydrolysis, indicating that MinE could displace MinC from the MinD-bilayer complex. In contrast, MinC was unable to displace MinE bound to the MinD-bilayer complex. These results suggest that MinE induces a conformational change in MinD bound to the bilayer that results in the release of MinC. Also, it is argued that binding of MinD to the membrane activates MinC.     

A basic region neighboring the lysine-rich C-terminus of protein SRP19 is required for binding to signal recognition particle RNA.

To identify some of the determinants in the 19-kilodalton protein of signal recognition particle (SRP19) for binding to signal recognition particle RNA, two mutant derivatives of the SRP19 were constructed, lacking 14 and 24 C-terminal amino acids. Polypeptides were transcribed and translated in vitro and tested for their ability to bind to signal recognition particle RNA by retention of protein-RNA complexes on DEAE-Sepharose. Both mutant polypeptides form complexes with the RNA, demonstrating that the 24 C-terminal amino acids, which include a lysine-rich sequence at positions 136-144, are dispensable. A third mutant polypeptide, in which eight additional amino acids were removed by oligonucleotide-directed digestion of the mRNA, was unable to bind. The amino acids in the sequence PKLKTRTQ correspond to positions 113-120; they are suggested to be involved in interaction with signal recognition particle RNA.     

ARF6 is required for growth factor- and rac-mediated membrane ruffling in macrophages at a stage distal to rac membrane targeting

Activation of Rac1, a member of the Rho family of GTPases, is associated with multiple cellular responses, including membrane ruffling and focal complex formation. The mechanisms by which Rac1 is coupled to these functional responses are not well understood. It was recently shown that ARF6, a GTPase implicated in cytoskeletal alterations and a membrane recycling pathway, is required for Rac1-dependent phagocytosis in macrophages (Q. Zhang et al., J. Biol. Chem. 273:19977-19981, 1998). To determine whether ARF6 is required for Rac1-dependent cytoskeletal responses in macrophages, we expressed wild-type (WT) or guanine nucleotide binding-deficient alleles (T27N) of ARF6 in macrophages coexpressing activated alleles of Rac1 (Q61L) or Cdc42 (Q61L) or stimulated with colony-stimulating factor 1 (CSF-1). Expression of ARF6 T27N but not ARF6 WT inhibited ruffles mediated by Rac1 Q61L or CSF-1. In contrast, expression of ARF6 T27N did not inhibit Rac1 Q61L-mediated focal complex formation and did not impair Cdc42 Q61L-mediated filopodial formation. Cryoimmunogold electron microscopy demonstrated the presence of ARF6 in membrane ruffles induced by either CSF-1 or Rac1 Q61L. Addition of CSF-1 to macrophages led to the redistribution of ARF6 from the interior of the cell to the plasma membrane, suggesting that this growth factor triggers ARF6 activation. Direct targeting of Rac1 to the plasma membrane did not bypass the blockade in ruffling induced by ARF6 T27N, indicating that ARF6 regulates a pathway leading to membrane ruffling that occurs after the activation and membrane association of Rac. These data demonstrate that intact ARF6 function is required for coupling activated Rac to one of several effector pathways and suggest that a principal function of ARF6 is to coordinate Rac activation with plasma membrane-based protrusive events.     

The MinD protein is a membrane ATPase required for the correct placement of the Escherichia coli division site.

The proper placement of the cell division site in Escherichia coli requires the site-specific inactivation of potential division sites at the cell poles in a process that is mediated by the MinC, MinD and MinE proteins. During the normal division cycle MinD plays two roles. It activates the MinC-dependent mechanism that is responsible for the inactivation of potential division sites and it also renders the division inhibition system sensitive to the topological specificity factor MinE. MinE suppresses the division block at the normal division site at mid-cell but not all cell poles, thereby ensuring the normal division pattern. In this study the MinD protein was purified to homogeneity and shown to bind ATP and to have ATPase activity. When the putative ATP binding domain of MinD was altered by site-directed mutagenesis, the mutant protein was no longer able to activate the MinC-dependent division inhibition system. Immunoelectron microscopy showed that MinD was located in the inner membrane region of the cell envelope. These results show that MinD is a membrane ATPase and suggest that the ATPase activity plays an essential role in the functions of the MinD protein during the normal division process.     

A conserved motif at the C terminus of sororin is required for sister chromatid cohesion

Sororin is a positive regulator of sister chromatid cohesion that interacts with the cohesin complex. Sororin is required for the increased stability of the cohesin complex on chromatin following DNA replication and sister chromatid cohesion during G(2). The mechanism by which sororin ensures cohesion is currently unknown. Because the primary sequence of sororin does not contain any previously characterized structural or functional motifs, we have undertaken a structure-function analysis of the sororin protein. Using a series of mutant derivatives of sororin, we show that the ability of sororin to bind to chromatin is separable from both its role in sister chromatid cohesion and its interaction with the cohesin complex. We also show that derivatives of sororin with deletions or mutations in the conserved C terminus fail to rescue the loss-of-cohesion phenotype caused by sororin RNAi and that these mutations also abrogate the association of sororin with the cohesin complex. Our data suggest that the interaction of the highly conserved motif at the C terminus of sororin with the cohesin complex is critical to its ability to mediate sister chromatid cohesion.     

The conserved C-terminal tail of FtsZ is required for the septal localization and division inhibitory activity of MinCC/MinD

The Escherichia coli Min system contributes to spatial regulation of cytokinesis by preventing assembly of the Z ring away from midcell. MinC is a cell division inhibitor whose activity is spatially regulated by MinD and MinE. MinC has two functional domains of similar size, both of which have division inhibitory activity in the proper context. However, the molecular mechanism of the inhibitory action of either domain is not very clear. Here, we report that the septal localization and division inhibitory activity of MinC^C/MinD requires the conserved C-terminal tail of FtsZ. This tail also mediates interaction with two essential division proteins, ZipA and FtsA, to link FtsZ polymers to the membrane. Overproduction of MinC^C/MinD displaces FtsA from the Z ring and eventually disrupts the Z ring, probably because it also displaces ZipA. These results support a model for the division inhibitory action of MinC/MinD. MinC/MinD binds to ZipA and FtsA decorated FtsZ polymers located at the membrane through the MinC^C/MinD–FtsZ interaction. This binding displaces FtsA and/or ZipA, and more importantly, positions MinC^N near the FtsZ polymers making it a more effective inhibitor.     

A conserved proliferating cell nuclear antigen-interacting protein sequence in Chk1 is required for checkpoint function

Human checkpoint kinase 1 (Chk1) is an essential kinase required for cell cycle checkpoints and for coordination of DNA synthesis. To gain insight into the mechanisms by which Chk1 carries out these functions, we used mass spectrometry to identify previously uncharacterized interacting partners of Chk1. We describe a novel interaction between Chk1 and proliferating cell nuclear antigen (PCNA), an essential component of the replication machinery. Binding between Chk1 and PCNA was reduced in the presence of hydroxyurea, suggesting that the interaction is regulated by replication stress. A highly conserved PCNA-interacting protein (PIP) box motif was identified in Chk1. The intact PIP box is required for efficient DNA damage-induced phosphorylation and release of activated Chk1 from chromatin. We find that the PIP box of Chk1 is crucial for Chk1-mediated S-M and G(2)-M checkpoint responses. In addition, we show that mutations in the PIP box of Chk1 lead to decreased rates of replication fork progression and increased aberrant replication. These findings suggest an additional mechanism by which essential components of the DNA replication machinery interact with the replication checkpoint apparatus.     

Compensation for the loss of the conserved membrane targeting sequence of FtsA provides new insights into its function

The bacterial actin homologue FtsA has a conserved C-terminal membrane targeting sequence (MTS). Deletion or point mutations in the MTS, such as W408E, were shown previously to inactivate FtsA function and inhibit cell division. Because FtsA binds to the tubulin-like FtsZ protein that forms the Z ring, it is thought that the MTS of FtsA is required, along with the transmembrane protein ZipA, to assemble the Z ring and anchor it to the cytoplasmic membrane. Here, we show that despite its reduced membrane binding, FtsA-W408E could localize to the Z ring and recruit the late cell division protein FtsI, but was defective in self-interaction and recruitment of FtsN, another late cell division protein. These defects could be suppressed by a mutation that stimulates membrane association of FtsA-W408E, or by expressing a tandem FtsA-W408E. Remarkably, the FtsA MTS could be completely replaced with the transmembrane domain of MalF and remain functional for cell division. We propose that FtsA function in cell division depends on additive effects of membrane binding and self-interaction, and that the specific requirement of an amphipathic helix for tethering FtsA to the membrane can be bypassed.     

MinD and role of the deviant Walker A motif,dimerization and membrane binding in oscillation

The ATPase activity of MinD is required for it to oscillate between the ends of the cell and spatially regulate cell division in Escherichia coli. It is a member of a functionally diverse subgroup of ATPases which are involved in activities ranging from nitrogen fixation (NifH) to plasmid segregation (ParA). All members of the subgroup have a deviant Walker A motif which contains a conserved 'signature' lysine that characterizes this subgroup. In the NifH homodimer the signature lysines make intermonomer contact with the bound nucleotides indicating a role in ATP hydrolysis. ATP binding to NifH leads to formation of an active dimer that associates with a partner that is also a dimer. Because ATP hydrolysis is coupled to formation of the complex, the complex is only transient. In the presence of ATP MinD binds MinC and goes to the membrane, however, the ATPase is not stimulated and the complex is stable. Subsequent interaction of this complex with MinE, however, leads to ATPase stimulation and release of the Min proteins from the membrane. The sequential interaction of MinD with these two proteins, which is dictated by the membrane, is critical to the oscillatory mechanism involved in spatial regulation of division.