High ATPase Activity Research Articles

Elevated VCP ATPase Activity Correlates With Disease Onset in Multisystem Proteinopathy-1.

Multisystem proteinopathy-1 (MSP1) is a late onset disease with >50 pathogenic variants in p97/VCP. MSP1 patients have multiple phenotypes that include inclusion body myopathy, Paget disease of the bone, amyotrophic lateral sclerosis, and frontotemporal dementia. There have been no clear genotype-phenotype correlations. We sought to identify genotype-phenotype correlations and associate these with VCP intrinsic ATPase activity. Patients with MSP1 were identified from the literature and the Cure VCP patient registry. Age at onset and at loss of ambulation were collated. VCP intrinsic ATPase activity was evaluated from recombinant purified protein. Among the 5 most common pathogenic VCP variants in MSP1 patients, R155C patients had the earliest average age at onset (38.15±9.78). This correlated with higher ATPase activity. Evaluation of 5 variants confirmed an inverse correlation between age at onset and ATPase activity (r = -0.94, p = 0.01). Previous studies have reported that VCP pathogenic variants are "hyperactive." Whether this elevation in VCP ATPase activity is relevant to disease is unclear. Our study supports that in vitro VCP activity correlates with disease onset and may guide the prognosis of patients with rare or unreported variants. Moreover, it suggests that inhibition of VCP ATPase activity in MSP1 may be therapeutic.

Relationship between jejunum ATPase activity and antioxidant function on the growth performance, feed conversion efficiency, and jejunum microbiota in Hu sheep (Ovis aries)

BackgroundATPase activity and the antioxidant function of intestinal tissue can reflect intestinal cell metabolic activity and oxidative damage, which might be related to intestinal function. However, the specific influence of intestinal ATPase activity and antioxidant function on growth performance, feed conversion efficiency, and the intestinal microbiota in sheep remains unclear.ResultsThis study analyzed the correlation between ATPase activity and antioxidant function in the jejunum of 92 Hu sheep and their growth performance and feed conversion efficiency. Additionally, individuals with the highest (H group) and lowest (L group) jejunum MDA content and Na+ K+-ATPase activity were further screened, and the effects of jejunum ATPase activity and MDA content on the morphology and microbial community of sheep intestines were analyzed. There was a significant correlation between jejunum ATPase and SOD activity and the initial weight of Hu sheep (P < 0.01). The H-MDA group exhibited significantly higher average daily gain (ADG) from 0 to 80 days old and higher body weight (BW) after 80 days. ATPase and SOD activities, and MDA levels correlated significantly and positively with heart weight. The jejunum crypt depth and circular muscle thickness in the H-ATP group were significantly higher than in the L-ATP group, and the villus length, crypt depth, and longitudinal muscle thickness in the H-MDA group were significantly higher than in the L-MDA group (P < 0.01). High ATPase activity and MDA content significantly reduced the jejunum microbial diversity, as indicated by the Chao1 index and observed species, and affected the relative abundance of specific taxa. Among species, the relative abundance of Olsenella umbonata was significantly higher in the H-MDA group than in the L-MDA group (P < 0.05), while Methanobrevibacter ruminantium abundance was significantly lower than in the L-MDA group (P < 0.05). In vitro culture experiments confirmed that MDA promoted the proliferation of Olsenella umbonata. Thus, ATPase and SOD activities in the jejunum tissues of Hu sheep are predominantly influenced by congenital factors, and lambs with higher birth weights exhibit lower Na+ K+-ATPase, Ca2+ Mg2+-ATPase, and SOD activities.ConclusionsThe ATPase activity and antioxidant performance of intestinal tissue are closely related to growth performance, heart development, and intestinal tissue morphology. High ATPase activity and MDA content reduced the microbial diversity of intestinal tissue and affect the relative abundance of specific taxa, representing a potential interaction between the host and its intestinal microbiota.

Regulation of the cell division hydrolase RipC by the FtsEX system in Mycobacterium tuberculosis

The FtsEX complex regulates, directly or via a protein mediator depending on bacterial genera, peptidoglycan degradation for cell division. In mycobacteria and Gram-positive bacteria, the FtsEX system directly activates peptidoglycan-hydrolases by a mechanism that remains unclear. Here we report our investigation of Mycobacterium tuberculosis FtsEX as a non-canonical regulator with high basal ATPase activity. The cryo-EM structures of the FtsEX system alone and in complex with RipC, as well as the ATP-activated state, unveil detailed information on the signal transduction mechanism, leading to the activation of RipC. Our findings indicate that RipC is recognized through a “Match and Fit” mechanism, resulting in an asymmetric rearrangement of the extracellular domains of FtsX and a unique inclined binding mode of RipC. This study provides insights into the molecular mechanisms of FtsEX and RipC regulation in the context of a critical human pathogen, guiding the design of drugs targeting peptidoglycan remodeling.

The lysosomal transporter TAPL has a dual role as peptide translocator and phosphatidylserine floppase

TAPL is a lysosomal ATP-binding cassette transporter that translocates a broad spectrum of polypeptides from the cytoplasm into the lysosomal lumen. Here we report that, in addition to its well-known role as a peptide translocator, TAPL exhibits an ATP-dependent phosphatidylserine floppase activity that is the possible cause of its high basal ATPase activity and of the lack of coupling between ATP hydrolysis and peptide efflux. We also present the cryo-EM structures of mouse TAPL complexed with (i) phospholipid, (ii) cholesteryl hemisuccinate (CHS) and 9-mer peptide, and (iii) ADP·BeF3. The inward-facing structure reveals that F449 protrudes into the cylindrical transport pathway and divides it into a large hydrophilic central cavity and a sizable hydrophobic upper cavity. In the structure, the peptide binds to TAPL in horizontally-stretched fashion within the central cavity, while lipid molecules plug vertically into the upper cavity. Together, our results suggest that TAPL uses different mechanisms to function as a peptide translocase and a phosphatidylserine floppase.

Comparison between two cultivars of longan fruit cv. ‘Dongbi’ and ‘Fuyan’ in the metabolisms of lipid and energy and its relation to pulp breakdown

This work studied the difference in pulp breakdown between two cultivars of longan cv. ‘Dongbi’ and ‘Fuyan’ from the aspect of metabolisms of lipid and energy. The results reflected that, compared to ‘Fuyan’ longan, ‘Dongbi’ longan had higher levels of energy charge, U/S and IUFA, and higher amounts of USFA, PC, PI, ATP and ADP. Moreover, ‘Dongbi’ longan exhibited lower levels of SFA, PA, AMP and cell membrane permeability. Also, lower PLD, LOX and lipase activities, but higher ATPase activity, lower pulp breakdown index, and better pulp appearance were exhibited in ‘Dongbi’ longan. These data revealed that the mitigated pulp breakdown in ‘Dongbi’ longan was due to the comprehensive coordination of metabolisms in lipid and energy through maintaining a higher level of energy, a higher unsaturation degree of fatty acids, delaying the degradation of phospholipids, and better retaining the membrane structural integrity of microsome and entire cell.

Enhanced survival of fluidized bed-dried microencapsulated Saccharomyces cerevisiae cells in the presence of Hongqu rice distiller's grain peptides

Saccharomyces cerevisiae (strain JH301) cells were micro-encapsulated in microcrystalline cellulose and Hongqu rice distiller's grain peptides (HRP) and subjected to fluidized bed drying. The effect of HRP on the viability of yeast in response to thermal drying was investigated. The moisture-viability profiles showed that HRP reduced the drying rate of yeast cells and increased viability in a concentration-dependent manner. Scanning electron microscopy revealed that HRP protected the morphology of the yeast cells, which presented a normal smooth structure and compact shape after thermal drying. The results of low-field 1H nuclear magnetic resonance and infrared spectroscopy revealed that HRP controls the binding water of yeast powder as opposed to interacting with the membrane surface proteins, further reducing the damage to phospholipids in the cell membrane during drying. Flow cytometry and confocal laser scanning microscopy confirmed that 3% HRP could reduce the proportion of membrane-damaged cells by nearly 19%. After drying, the proportion of unsaturated fatty acids in the cell membrane of JH301-3% HRP was 10.36% higher than that of JH301 in the absence of HRP, achieving increased membrane integrity and higher ATPase activity after rehydration.

Plasma Membrane Ca2+ Pump PMCA4z Is More Active Than Splicing Variant PMCA4x.

The plasma membrane Ca2+ pumps (PMCA) are P-ATPases that control Ca2+ signaling and homeostasis by transporting Ca2+ out of the eukaryotic cell. Humans have four genes that code for PMCA isoforms (PMCA1-4). A large diversity of PMCA isoforms is generated by alternative mRNA splicing at sites A and C. The different PMCA isoforms are expressed in a cell-type and developmental-specific manner and exhibit differential sensitivity to a great number of regulatory mechanisms. PMCA4 has two A splice variants, the forms “x” and “z”. While PMCA4x is ubiquitously expressed and relatively well-studied, PMCA4z is less characterized and its expression is restricted to some tissues such as the brain and heart muscle. PMCA4z lacks a stretch of 12 amino acids in the so-called A-M3 linker, a conformation-sensitive region of the molecule connecting the actuator domain (A) with the third transmembrane segment (M3). We expressed in yeast PMCA4 variants “x” and “z”, maintaining constant the most frequent splice variant “b” at the C-terminal end, and obtained purified preparations of both proteins. In the basal autoinhibited state, PMCA4zb showed a higher ATPase activity and a higher apparent Ca2+ affinity than PMCA4xb. Both isoforms were stimulated by calmodulin but PMCA4zb was more strongly activated by acidic lipids than PMCA4xb. The results indicate that a PMCA4 intrinsically more active and more responsive to acidic lipids is produced by the variant “z” of the splicing site A.

Inactivation of mitochondrial complex I stimulates chloroplast ATPase inPhyscomitrium patens

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Intermediates in allosteric equilibria of DnaK–ATP interactions with substrate peptides

Hsp70 molecular chaperones facilitate protein disaggregation and proper folding through iterative cycles of polypeptide binding and release that are allosterically coupled to ATP binding and hydrolysis. Hsp70s are ubiquitous and highly conserved across all of life; they bind ATP at an N-terminal nucleotide-binding domain (NBD) and client peptides in the substrate-binding domain (SBD). The NBD and SBD are connected by a highly conserved linker segment that is integrated into the NBD when ATP is bound but is flexible when the NBD is nucleotide-free or bound with ADP. Allosteric coupling is lost when the linker is flexible, and the freed SBD binds peptide clients with high affinity. It was recently discovered that Hsp70-ATP is in an equilibrium between a restraining state (R) with little affinity for peptides and a low ATPase activity, and a stimulating state (S) that binds peptides efficiently, but with rapid kinetics, and has a relatively high ATPase activity. While attempting to characterize the S state, crystal structures of DnaK-ATP were obtained that demonstrate intrinsic Hsp70 plasticity that affects binding interactions with substrate peptides. These structures provide insights into intermediate states along transition pathways in the Hsp70 chaperone cycle.

Basic mechanism of the autonomous ClpG disaggregase

Bacterial survival during lethal heat stress relies on the cellular ability to reactivate aggregated proteins. This activity is typically executed by the canonical 70-kDa heat shock protein (Hsp70)–ClpB bichaperone disaggregase, which is most widespread in bacteria. The ClpB disaggregase is a member of the ATPase associated with diverse cellular activities protein family and exhibits an ATP-driven threading activity. Substrate binding and stimulation of ATP hydrolysis depends on the Hsp70 partner, which initiates the disaggregation reaction. Recently elevated heat resistance in gamma-proteobacterial species was shown to be mediated by the ATPase associated with diverse cellular activities protein ClpG as an alternative disaggregase. Pseudomonas aeruginosa ClpG functions autonomously and does not cooperate with Hsp70 for substrate binding, enhanced ATPase activity, and disaggregation. With the underlying molecular basis largely unknown, the fundamental differences in ClpG- and ClpB-dependent disaggregation are reflected by the presence of sequence alterations and additional ClpG-specific domains. By analyzing the effects of mutants lacking ClpG-specific domains and harboring mutations in conserved motifs implicated in ATP hydrolysis and substrate threading, we show that the N-terminal, ClpG-specific N1 domain generally mediates protein aggregate binding as the molecular basis of autonomous disaggregation activity. Peptide substrate binding strongly stimulates ClpG ATPase activity by overriding repression by the N-terminal N1 and N2 domains. High ATPase activity requires two functional nucleotide binding domains and drives substrate threading which ultimately extracts polypeptides from the aggregate. ClpG ATPase and disaggregation activity is thereby directly controlled by substrate availability.

The effect of fish freshness on myosin denaturation in flounder Paralichthys olivaceus muscle during frozen storage

The effect of fish freshness on myosin denaturation during the frozen storage of flounder muscle was studied. The muscle of instantaneously killed flounder was immediately stored at − 20 °C (pre-rigor mortis; day 0), or after storage at 0 °C for 2 days (rigor mortis) or 5 days (post-rigor mortis). Myosin denaturation in frozen stored muscle was studied by measuring Ca2+-ATPase activity, myosin solubility in the presence of salt (salt solubility), and monomeric myosin content. High ATPase activity was maintained in the day-0 samples of instantaneously killed flounder during the early phase of frozen storage. High salt solubility of myosin was also observed in these samples. However, the beneficial effect of frozen storage was lost when the storage period exceeded 2 months. There was no difference in myosin denaturation among the three groups of samples after 2 months of frozen storage. The myosin denaturation profile during the frozen storage of muscle was characterized by high salt solubility in the presence of Mg-ATP, even after ATPase inactivation, especially in the day-0 samples. The salt-soluble fraction contained a large amount of aggregated myosin, as revealed by (NH4)2SO4 fractionation. The monomeric myosin content was explained by Ca2+-ATPase inactivation in the frozen stored meat for all three groups. This study demonstrated that the high quality of flounder meat is maintained during short-term frozen storage, but that the beneficial effects of frozen storage are lost over longer periods of time.

Functional analysis and classification of homozygous and hypomorphic ABCA4 variants associated with Stargardt macular degeneration.

Stargardt macular degeneration (Stargardt disease 1 [STGD1]) is caused by mutations in the gene encoding ABCA4, an ATP-binding cassette protein that transports N-retinylidene-phosphatidylethanolamine (N-Ret-PE) across photoreceptor membranes. Reduced ABCA4 activity results in retinoid accumulation leading to photoreceptor degeneration. The disease onset and severity vary from severe loss in visual acuity in the first decade to mild visual impairment late in life. We determined the effect of 22 disease-causing missense mutations on the expression and ATPase activity of ABCA4 in the absence and presence of N-Ret-PE. Three classes were identified that correlated with the disease onset in homozygous STGD1 individuals: Class 1 exhibited reduced ABCA4 expression and ATPase activity that was not stimulated by N-Ret-PE; individuals homozygous for these variants had an early disease onset (≤13years); Class 2 showed reduced ATPase activity with limited stimulation by N-Ret-PE; these correlated with moderate disease onset (14-40years); and Class 3 displayed high expression and ATPase activity that was strongly activated by N-Ret-PE; these were associated with late disease onset (>40years). On the basis of our results, we introduce a functionality index for gauging the effect of missense mutations on STGD1 severity. Our studies support the mild phenotype exhibited by the p.Gly863Ala, p.Asn1868Ile, and p.Gly863Ala/p.Asn1868Ile variants.

Transcriptome analysis reveals that the multiple metabolic pathways were related to gluten polymerization in different quality wheats (Triticum aestivum L.).

The rapid development of transcriptome sequencing technology has contributed to the discovery of numerous genes in plant; however, the role of gene expression in postharvest wheat remains largely unexplored. In this study, differentially expressed genes (DEGs) were identified by RNA‐seq in different quality wheats. The 102.6 Gb clean reads had been yielded from the nine RNA‐seq libraries. Typically, there were 1791 upregulated and 2,677 downregulated DEGs, respectively, in strong‐gluten wheat compared with weak‐gluten wheat. Specifically, a total of 4,468 DEGs were classified into 286 Gene Ontology (GO) terms and 131 Kyoto Encyclopedia of Genes and Genomes terms (KEGG). Moreover, the storage protein components, starch and sucrose metabolism, and plant hormone signal transduction‐related genes were discovered, which had involved 109 DEGs. The wet gluten proteins content was 35.24% and 17.36%, and the glutenin macropolymer content was 6.38% and 5.01% between the strong‐ and weak‐gluten wheat, respectively. The POD activities of the different quality wheats were 6,571.14, 5,341.24, and 4,851.48 U/g/min, respectively. The significant difference of starch and sucrose metabolism, hormone, POD, and CAT enzyme along with the higher ATPase activity might potentially affect gluten polymerization, which might thereby result in the different qualities of wheats.

Ablation of the N terminus of cardiac essential light chain promotes the super-relaxed state of myosin and counteracts hypercontractility in hypertrophic cardiomyopathy mutant mice.

In this study, we focus on the molecular mechanisms associated with the A57G (Ala57-to-Gly57) mutation in myosin essential light chains (ELCs), found to cause hypertrophic cardiomyopathy (HCM) in humans and in mice. Specifically, we studied the effects of A57G on the super-relaxed (SRX) state of myosin that may contribute to the hypercontractile cross-bridge behavior and ultimately lead to pathological cardiac remodeling in transgenic Tg-A57G mice. The disease model was compared to Tg-WT mice, expressing the wild-type human ventricular ELC, and analyzed against Tg-Δ43 mice, expressing the N-terminally truncated ELC, whose hearts hypertrophy with time but do not show any abnormalities in cardiac morphology or function. Our data suggest a new role for the N terminus of cardiac ELC (N-ELC) in modulation of myosin cross-bridge function in the healthy as well as in HCM myocardium. The lack of N-ELC in Tg-Δ43 mice was found to significantly stabilize the SRX state of myosin and increase the number of myosin heads occupying a low-energy state. In agreement, Δ43 hearts showed significantly decreased ATP utilization and low actin-activated myosin ATPase compared with A57G and WT hearts. The hypercontractile activity of A57G-ELC cross-bridges was manifested by the inhibition of the SRX state, increased number of myosin heads available for interaction with actin, and higher ATPase activity. Fiber mechanics studies, echocardiography examination, and assessment of fibrosis confirmed the development of two distinct forms of cardiac remodeling in these two ELC mouse models, with pathological cardiac hypertrophy in Tg-A57G, and near physiologic cardiac growth in Tg-Δ43 animals.

Interdomain communication suppressing high intrinsic ATPase activity of Sse1 is essential for its co-disaggregase activity with Ssa1.

In eukaryotes, Hsp110s are unambiguous cognates of the Hsp70 chaperones, in primary sequence, domain organization, and structure. Hsp110s function as nucleotide exchange factors (NEFs) for the Hsp70s although their apparent loss of Hsp70-like chaperone activity, nature of interdomain communication, and breadth of domain functions are still puzzling. Here, by combining single-molecule FRET, small angle X-ray scattering measurements (SAXS), and MD simulation, we show that yeast Hsp110, Sse1 lacks canonical Hsp70-like interdomain allostery. However, the protein exhibits unique noncanonical conformational changes within its domains. Sse1 maintains an open-lid substrate-binding domain (SBD) in close contact with its nucleotide-binding domain (NBD), irrespective of its ATP hydrolysis status. To further appreciate such ATP-hydrolysis-independent exhaustive interaction between two domains of Hsp110s, NBD-SBD chimera was constructed between Hsp110 (Sse1) and Hsp70 (Ssa1). In Sse1/Ssa1 chimera, we observed undocking of two domains leading to complete loss of NEF activity of Sse1. Interestingly, chimeric proteins exhibited significantly enhanced ATPase rate of Sse1-NBD compared to wild-type protein, implying that intrinsic ATPase activity of the protein remains mostly repressed. Apart from repressing the high ATPase activity of its NBD, interactions between two domains confer thermal stability to Sse1 and play critical role in the (co)chaperoning function of Sse1 in Ssa1-mediated disaggregation activity. Altogether, Sse1 exhibits a unique interdomain interaction, which is essential for its NEF activity, suppression of high intrinsic ATPase activity, co-chaperoning activity in disaggregase machinery, and stability of the protein.

Two-Step Activation Mechanism of the ClpB Disaggregase for Sequential Substrate Threading by the Main ATPase Motor

SummaryAAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.

Structural comparison of the vacuolar and Golgi V-ATPases from Saccharomyces cerevisiae

Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast Saccharomyces cerevisiae, isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that VO complexes containing Stv1p could be readily purified without attached V1 regions. We used this effect to determine structures of the membrane-embedded VO region with Stv1p at 3.1-Å resolution, which we compare with a structure of the VO region with Vph1p that we determine to 3.2-Å resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.

Conformational Dynamics in the Binding-Protein-Independent Mutant of the Escherichia coli Maltose Transporter, MalG511, and Its Interaction with Maltose Binding Protein.

MalG511 is a genetically selected binding-protein-independent mutant of the Escherichia coli maltose transporter MalFGK2, which retains specificity for maltose and shows a high basal ATPase activity in the absence of maltose binding protein (MBP). It shows an intriguing biphasic behavior in maltose transport assays in the presence of MBP, with low levels of MBP stimulating the activity and higher levels (>50 μM) inhibiting the transport activity. Remarkably, the rescuing effect of the MBP suppressor mutant, MBPG13D, turns it into an attractive model for studying regulatory mechanisms in the ABC transporter superfamily. It is hypothesized that the special characteristics of MalG511 result from mutations that shift its equilibrium toward the transition state of MalFGK2. We tested this hypothesis by using site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy, which showed conformational changes in MalG511 and its interaction with MBP and MBPG13D during its catalytic cycle. We found that MalG511 utilizes the same alternate access mechanism as MalFGK2, including all three open, semi-open, and closed states of the MalK dimer, to transport maltose across the membrane. However, the equilibrium of this mutant is shifted toward the semi-open state in its resting state and interacts with MBP with high affinity, providing an explanation for the inhibition of MalG511 by MBP at higher concentrations. In contrast, the mutant binding protein, MBPG13D, interacts with lower affinity and could restore MalG511 to a normal catalytic cycle.

Stand-alone ClpG disaggregase confers superior heat tolerance to bacteria

AAA+ disaggregases solubilize aggregated proteins and confer heat tolerance to cells. Their disaggregation activities crucially depend on partner proteins, which target the AAA+ disaggregases to protein aggregates while concurrently stimulating their ATPase activities. Here, we report on two potent ClpG disaggregase homologs acquired through horizontal gene transfer by the species Pseudomonas aeruginosa and subsequently abundant P. aeruginosa clone C. ClpG exhibits high, stand-alone disaggregation potential without involving any partner cooperation. Specific molecular features, including high basal ATPase activity, a unique aggregate binding domain, and almost exclusive expression in stationary phase distinguish ClpG from other AAA+ disaggregases. Consequently, ClpG largely contributes to heat tolerance of P. aeruginosa primarily in stationary phase and boosts heat resistance 100-fold when expressed in Escherichia coli This qualifies ClpG as a potential persistence and virulence factor in P. aeruginosa.

Fission yeast myosin Myo2 is down-regulated in actin affinity by light chain phosphorylation

Studies in fission yeast Schizosaccharomyces pombe have provided the basis for the most advanced models of the dynamics of the cytokinetic contractile ring. Myo2, a class-II myosin, is the major source of tension in the contractile ring, but how Myo2 is anchored and regulated to produce force is poorly understood. To enable more detailed biochemical/biophysical studies, Myo2 was expressed in the baculovirus/Sf9 insect cell system with its two native light chains, Rlc1 and Cdc4. Milligram yields of soluble, unphosphorylated Myo2 were obtained that exhibited high actin-activated ATPase activity and in vitro actin filament motility. The fission yeast specific chaperone Rng3 was thus not required for expression or activity. In contrast to nonmuscle myosins from animal cells that require phosphorylation of the regulatory light chain for activation, phosphorylation of Rlc1 markedly reduced the affinity of Myo2 for actin. Another unusual feature of Myo2 was that, unlike class-II myosins, which generally form bipolar filamentous structures, Myo2 showed no inclination to self-assemble at approximately physiological salt concentrations, as analyzed by sedimentation velocity ultracentrifugation. This lack of assembly supports the hypothesis that clusters of Myo2 depend on interactions at the cell cortex in structural units called nodes for force production during cytokinesis.