PilT mutations lead to simultaneous defects in competence for natural transformation and twitching motility in piliated Neisseria gonorrhoeae

Neisseria gonorrhoeae, the Gram-negative aetiological agent of gonorrhoea, is one of many mucosal pathogens of man that expresses competence for natural transformation. Expression of this phenotype by gonococci appears to rely on the expression of type IV pili (Tfp), but the mechanistic basis for this relationship remains unknown. During studies of gonococcal pilus biogenesis, a homologue of the PilT family of proteins, required for Tfp-dependent twitching motility in Pseudomonas aeruginosa and social gliding motility in Myxococcus xanthus, was discovered. Like the findings in these other species, we show here that gonococcal pilT mutants constructed in vitro no longer display twitching motility. In addition, we demonstrate that they have concurrently lost the ability to undergo natural transformation, despite the expression of structurally and morphologically normal Tfp. These results were confirmed by the findings that two classes of spontaneous mutants that failed to express twitching motility and transformability carried mutations in pilT. Piliated pilT mutants and a panel of pilus assembly mutants were found to be deficient in sequence-specific DNA uptake into the cell, the earliest demonstrable step in neisserial competence. The PilT-deficient strains represent the first genetically defined mutants that are defective in DNA uptake but retain Tfp expression.     

The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA

Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.     

Aquifex aeolicus PilT,homologue of a surface motility protein,is a thermostable oligomeric NTPase

Bacterial surface motility works by retraction of surface-attached type IV pili. This retraction requires the PilT protein, a member of a large family of putative NTPases from type II and IV secretion systems. In this study, the PilT homologue from the thermophilic eubacterium Aquifex aeolicus was cloned, overexpressed, and purified. A. aeolicus PilT was shown to be a thermostable ATPase with a specific activity of 15.7 nmol of ATP hydrolyzed/min/mg of protein. This activity was abolished when a conserved lysine in the nucleotide-binding motif was altered. The substrate specificity was low; UTP, CTP, ATP, GTP, dATP, and dGTP served as substrates, UTP having the highest activity of these in vitro. Based on sedimentation equilibrium and size exclusion chromatography, PilT was identified as a approximately equal 5- to 6-subunit oligomer. Potential implications of the NTPase activity of PilT in pilus retraction are discussed.     

SecA protein hydrolyzes ATP and is an essential component of the protein translocation ATPase of Escherichia coli.

Bacterial protein export requires two forms of energy input, ATP and the membrane electrochemical potential. Using an in vitro reaction reconstituted with purified soluble and peripheral membrane components, we can now directly measure the translocation-coupled hydrolysis of ATP. This translocation ATPase requires inner membrane vesicles, SecA protein and translocation-competent proOmpA. The stimulatory activity of membrane vesicles can be blocked by either antibody to the SecY protein or by preparing the membranes from a secY-thermosensitive strain which had been incubated at the non-permissive temperature in vivo. The SecA protein itself has more than one ATP binding site. 8-azido-ATP inactivates SecA for proOmpA translocation and for translocation ATPase, yet does not inhibit a low level of ATP hydrolysis inherent in the isolated SecA protein. These data show that the SecA protein has a central role in coupling the hydrolysis of ATP to the transfer of pre-secretory proteins across the membrane.     

SufC hydrolyzes ATP and interacts with SufB from Thermotoga maritima

Genetic experiments in bacteria have shown the suf operon is involved in iron homeostasis and the oxidative stress response. The sufB and sufC genes that always occur together in bacteria are also found in plants, and even the malaria parasite, associated with the plastid organelle. Although the suf operon is believed to encode an iron-dependent ABC-transporter there is no direct evidence. By immunolocalization we show here that SufB and SufC are associated with the membrane of Escherichia coli. We also present kinetic studies with a recombinant version of SufC from Thermotoga maritima that shows it is an ATPase and that it interacts with SufB in vitro.     

A cAMP receptor protein, SYCRP1, is responsible for the cell motility of Synechocystis sp. PCC 6803

Disruption of the sycrp1 gene encoding a cyanobacterial cAMP receptor protein makes cells of Synechocystis sp. PCC 6803 non-motile. Electron microscopy showed that the sycrp1-deficient strain had a reduced number of thick pili on the cell surface compared with the wild-type strain. It is suggested that cAMP-SYCRP1 complex controls the biogenesis of pili.     

Evidence that the 116 kDa component of kinesin binds and hydrolyzes ATP

Kinesin was prepared from bovine brain as described previously for studies of translocation. A major component of kinesin, (116 kDa) was shown to undergo specific photocrosslinking with [alpha-32P]ATP, indicating it was an ATP-binding polypeptide. A low ATPase activity associated with kinesin was stimulated up to 5-fold by microtubules to a specific activity of 14 nmol . min-1 . mg-1. N-Ethylmaleimide inhibited both [alpha-32P]ATP binding to the 116 kDa polypeptide and microtubule-stimulated ATPase activity, suggesting that the 116 kDa polypeptide was the catalytic subunit of kinesin. Though the ATPase activity associated with kinesin is low, it may be sufficient to support motility assuming it is coupled to the velocity of translocation.     

Circadian autodephosphorylation of cyanobacterial clock protein KaiC occurs via formation of ATP as intermediate

The cyanobacterial circadian oscillator can be reconstituted in vitro; mixing three clock proteins (KaiA, KaiB, and KaiC) with ATP results in an oscillation of KaiC phosphorylation with a periodicity of ~24 h. The hexameric ATPase KaiC hydrolyzes ATP bound at subunit interfaces. KaiC also exhibits autokinase and autophosphatase activities, the latter of which is particularly noteworthy because KaiC is phylogenetically distinct from typical protein phosphatases. To examine this activity, we performed autodephosphorylation assays using (32)P-labeled KaiC. The residual radioactive ATP bound to subunit interfaces was removed using a newly established method, which included the dissociation of KaiC hexamers into monomers and the reconstitution of KaiC hexamers with nonradioactive ATP. This approach ensured that only the signals derived from (32)P-labeled KaiC were examined. We detected the transient formation of [(32)P]ATP preceding the accumulation of (32)P(i). Together with kinetic analyses, our data demonstrate that KaiC undergoes dephosphorylation via a mechanism that differs from those of conventional protein phosphatases. A phosphate group at a phosphorylation site is first transferred to KaiC-bound ADP to form ATP as an intermediate, which can be regarded as a reversal of the autophosphorylation reaction. Subsequently, the ATP molecule is hydrolyzed to form P(i). We propose that the ATPase active site mediates not only ATP hydrolysis but also the bidirectional transfer of the phosphate between phosphorylation sites and the KaiC-bound nucleotide. On the basis of these findings, we can now dissect the dynamics of the KaiC phosphorylation cycle relative to ATPase activity.     

PilT2 enhances the speed of gonococcal type IV pilus retraction and of twitching motility

Type IV pilus (T4P) dynamics is important for various bacterial functions including host cell interaction, surface motility, and horizontal gene transfer. T4P retract rapidly by depolymerization, generating large mechanical force. The gene that encodes the pilus retraction ATPase PilT has multiple paralogues, whose number varies between different bacterial species, but their role in regulating physical parameters of T4P dynamics remains unclear. Here, we address this question in the human pathogen Neisseria gonorrhoeae, which possesses two pilT paralogues, namely pilT2 and pilU. We show that the speed of twitching motility is strongly reduced in a pilT2 deletion mutant, while directional persistence time and sensitivity of speed to oxygen are unaffected. Using laser tweezers, we found that the speed of single T4P retraction was reduced by a factor of ≈ 2 in a pilT2 deletion strain, whereas pilU deletion showed a minor effect. The maximum force and the probability for switching from retraction to elongation under application of high force were not significantly affected. We conclude that the physical parameters of T4P are fine‐tuned through PilT2.     

ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts

Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.     

The relationship between ATP content and motility of bovine spermatozoa

The ATP content of bovine semen was measured using a bioluminescent technique. No ATP was detected in seminal plasma or diluent. The mean concentration of ATP (+/- sd) in ejaculated semen was 76.5+/-38.21 n moles ATP/10(8) spermatozoa and in diluted, frozen and thawed semen 10.00+/-4.74 n moles ATP/10(8) spermatozoa. The observed motilities of ejaculated and of diluted, frozen and thawed semen were linearly related to the ATP content r=0.652; p<0.01 and r=0.466; p<0.001 respectively). The mean ATP content and motility calculated for each bull were similarly related (r=0.857; p<0.02 and r=0.607; p<0.05). The technique shows promise in providing an objective measure of spermatozoan activity.     

A novel role for IQGAP1 protein in cell motility through cell retraction

IQGAP1 has emerged as a key component in the regulation of cytoskeleton dynamics during cell migration, maintenance of adherens junctions, microbial pathogenesis and intracellular trafficking. IQGAP1 is known to localize to the protruding edge of lamellipodia in a variety of cell types and interact with regulators of actin dynamics. Here, we provide evidence suggesting a novel role of IQGAP1 in cell motility through cell edge retraction. In some of the cell lines examined, IQGAP1 was markedly separated from WAVE localization suggesting IQGAP1 may localize to retracting edges. B16F10 mouse melanoma cells exhibited the most restricted separation in which the appearance of GFP-IQGAP1 correlated with cell edge retraction velocity and the disappearance of mCherry-Arp3. These results demonstrate that in some cell types IQGAP1 may function to promote cell retraction not lamellipodium edge protrusion. In addition, we examined co-localization of IQGAP1 with adhesion site markers, myosin IIA, calmodulin and IQGAP2. In areas rich in IQGAP1 there was decreased immunofluorescence staining of vinculin, paxillin and phosphorylated-tyrosine indicating adhesion site disassembly. Interestingly, calmodulin, but not myosin IIA or IQGAP2, co-localized with IQGAP1 in areas of cell retraction. Overall these results suggest a new role of IQGAP1, distinct form IQGAP2, in cell migration through up regulation of contractility and downregulation of adhesion sites potentially through calmodulin interaction.     

Function, structure and regulation of cyanobacterial and chloroplast ATP synthase

The proton-translocating ATP synthase from chloroplasts and cyanobacteria forms ATP upon photosynthetic electron transport by using the proton gradient across the thylakoid membrane. Both enzymes contain nine different subunits and from the similarity in gene organisation and the high degree of amino acid sequence homology of the subunits it appears that these ATP synthases might have a common ancestor. Both enzymes need to be activated by membrane energisation in order to perform catalytic activity but, in contrast to the chloroplast ATP synthase, that from the studied cyanobacteria (with the exception of Spirulina platensis ) shows no effect of the redox state on activation. Functionally, the cyanobacterial enzyme corresponds to the reduced form of the chloroplast ATP synthase. In the chloroplast enzyme a stretch of 9 amino acids, including two cysteines in the γ-subunit, is involved in this redox effect and this stretch is absent in cyanobacteria. With γ-mutants from the cyanobacterium Synechocystis 6803 the role of this stretch is studied. When active, both the cyanobacterial and the reduced chloroplast ATP synthase transport 4 protons per ATP synthesised and hydrolysed. This ratio may depend on the environment of the enzyme such as protein and lipid composition and pH.     

Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility

PilT is a hexameric ATPase required for bacterial type IV pilus retraction and surface motility. Crystal structures of ADP- and ATP-bound Aquifex aeolicus PilT at 2.8 and 3.2 A resolution show N-terminal PAS-like and C-terminal RecA-like ATPase domains followed by a set of short C-terminal helices. The hexamer is formed by extensive polar subunit interactions between the ATPase core of one monomer and the N-terminal domain of the next. An additional structure captures a nonsymmetric PilT hexamer in which approach of invariant arginines from two subunits to the bound nucleotide forms an enzymatically competent active site. A panel of pilT mutations highlights the importance of the arginines, the PAS-like domain, the polar subunit interface, and the C-terminal helices for retraction. We present a model for ATP binding leading to dramatic PilT domain motions, engagement of the arginine wire, and subunit communication in this hexameric motor. Our conclusions apply to the entire type II/IV secretion ATPase family.     

The clathrin adaptor complex responsible for somatodendritic protein sorting

Neuronal proteins contain "address labels" that govern their localization. In this issue of Neuron, Farías et?al. (2012) identify the machinery that recognizes one class of dendritic localization signals and establish its role in the polarization of dendritic proteins, including several postsynaptic receptors.