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The majority of mitochondrial proteins are nuclear encoded and imported into mitochondria as precursor proteins via dedicated translocases. The translocase of the inner membrane 22 (TIM22) is a multisubunit molecular machine specialized for the translocation of hydrophobic, multi‐transmembrane‐spanning proteins with internal targeting signals into the inner mitochondrial membrane. Here, we undertook a crosslinking‐mass spectrometry (XL‐MS) approach to determine the molecular arrangement of subunits of the human TIM22 complex. Crosslinking of the isolated TIM22 complex using the BS3 crosslinker resulted in the broad generation of crosslinks across the majority of TIM22 components, including the small TIM chaperone complex. The crosslinking data uncovered several unexpected features, opening new avenues for a deeper investigation into the steps required for TIM22‐mediated translocation in humans.

The ability of Mycobacteria to overcome oxidative stress is of paramount importance for its survival within the host. One of the key enzymes that are involved in protecting the bacterium from reactive oxygen species is the catalase-peroxidase (KatG). However, in strains resistant to the antibiotic isoniazid, KatG is rendered ineffective, which is associated with an increased expression of alkylhydroperoxide reductase subunit C (AhpC). Mycobacterial AhpC possesses a unique helical displacement when compared to its bacterial counterparts. Here, via mutagenesis studies, we demonstrate the importance of this helix for redox modulation of the catalytic activity of AhpC. Along with structural insights from crystallographic data, the impact of critical residues on the structure and flexibility of the helix and on AhpC oligomerization is described. Ministry of Education (MOE) Nanyang Technological University This study was supported by the AcademicResearch Fund (AcRF) Tier 1 ID889 Ministry of Edu-cation, Singapore, to GG, SMSC is grateful to receivean NTU Research Scholarship at Nanyang Technolog-ical University, Singapore.

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (kcat ≈ 104 s-1 ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine β-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).

Recently, understanding of protein targeting to the endoplasmic reticulum (ER) was expanded by the discovery of multiple pathways that function in parallel to the signal recognition particle (SRP). Guided entry of tail-anchored proteins and SRP independent (SND) are two such targeting pathways described in yeast. So far, no human SND component is functionally characterized. Here, we report hSnd2 as the first constituent of the human SND pathway able to support substrate-specific protein targeting to the ER. Similar to its yeast counterpart, hSnd2 is assumed to function as a membrane-bound receptor preferentially targeting precursors carrying C-terminal transmembrane domains. Our genetic and physical interaction studies show that hSnd2 is part of a complex network of targeting and translocation that is dynamically regulated.

We recently revealed that positively charged amino acids of Psb31, an extrinsic subunit found in diatom photosystem II (PSII), are involved in electrostatic interactions with PSII intrinsic subunits. However, the molecular interactions of Psb31 with PSII remain unclear. Here, we report the functional contribution of Lys residues in the binding of Psb31 to PSII using site-directed mutants of Psb31. Each of the K33A, K39A, K54A, K56A, K57A, and K69A mutants exhibits decreased binding affinities to PSII concomitantly with decreases in the O2 evolution activity. Conversely, each of the K24A, K76A, K80A, and K117A mutants functionally binds to PSII in a manner similar to wild-type Psb31. These results provide evidence that some Lys residues of Psb31 are responsible for electrostatic interactions with PSII.

Enteroviruses are responsible for a multitude of human diseases. Expansion of the virus capsid is associated with a cascade of conformational changes that allow the subsequent release of RNA. For the first time, this study presents a comprehensive bioinformatic screen for the prediction of interacting motifs within intraprotomer interfaces and across respective interfaces surrounding the five-fold and two-fold axes. The results identify a network of conserved motif residues involved in interactions in Enteroviruses that may be critical to capsid stabilisation, providing guidelines towards developing antivirals that interfere with viral expansion during RNA release. This article is protected by copyright. All rights reserved.

Salmonella enterica serovar Typhimurium has the ability to use molecular hydrogen as a respiratory electron donor. This is facilitated by three [NiFe]‐hydrogenases termed Hyd‐1, Hyd‐2, and Hyd‐5. Hyd‐1 and Hyd‐5 are homologous oxygen‐tolerant [NiFe]‐hydrogenases. A critical step in the biosynthesis of a [NiFe]‐hydrogenase is the proteolytic processing of the catalytic subunit. In this work, the role of the maturation protease encoded within the Hyd‐5 operon, HydD, was found to be partially complemented by the maturation protease encoded in the Hyd‐1 operon, HyaD. In addition, both maturation proteases were shown to form stable complexes, in vivo and in vitro, with the catalytic subunit of Hyd‐5. The protein–protein interactions were not detectable in a strain that could not make the enzyme metallocofactor.

Human mitochondrial complex I is the first enzyme of the mitochondrial respiratory chain. Complex I is composed of 45 subunits, seven encoded by mitochondrial DNA, while the remainder are encoded by nuclear DNA. All nuclear-encoded subunits are thought to be expressed as a single isoform. Here we reveal subunit NDUFV3 to be present in both the canonical 10 kDa and a novel 50 kDa isoform, generated through alternative splicing. Both isoforms assemble into complex I and their levels vary in different tissues. While the 50 kDa isoform appears to be dominant in HEK293T cells, we find either isoform alone is sufficient for assembly of mature complex I. NDUFV3 represents the first known complex I subunit present in two functional isoforms.

The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α2 β2 γ2 ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.

Potassium channels such as Kv2.1 are targeted to specific subcellular compartments to fulfill various functions. However, the mechanisms for their localization are poorly understood. Here, we show that KVS-4/Kv2.1 somatodendritic localization in Caenorhabditis elegansDA9 neuron requires UNC-101(AP-1 μ subunit). We define a bipartite sorting signal within KVS-4 consisting of a C-terminal EQMIL and N-terminal WNIIE motifs. The bipartite signal is sufficient to target nonpolarized transmembrane protein MIG-13 into DA9 somatodendritic compartments. Furthermore, we found that AP-1 interacts with the bipartite signal through UNC-101/AP-1 μ N-terminal predicted Longin-like domain. Our results provide new insight into the mechanisms of Kv2.1 post-Golgi sorting and targeting.

Phototropin (phot), a blue light receptor in plants, is composed of several domains: LOV1, LOV2, and a serine/threonine kinase (STK). LOV2 is the main regulator of light activation of STK. However, the detailed mechanism remains unclear. In this report, we focused on the linker region between LOV2 and STK excluding the Jα-helix. Spectroscopy and a kinase assay for the substituents in the linker region of Arabidopsis phot1 LOV2-STK indicated that the linker is involved in the activation of STK. A putative module in the middle of the linker would be critical for intramolecular signaling and/or regulation of STK.

Lytic polysaccharide monooxygenases (LPMOs) boost enzymatic depolymerization of recalcitrant polysaccharides, such as chitin and cellulose. We have studied a chitin-active LPMO domain (JdLPMO10A) that is considerably smaller (15.5 kDa) than all structurally characterized LPMOs so far and that is part of a modular protein containing a GH18 chitinase. The 1.55 Å resolution structure revealed deletions of interacting loops that protrude from the core β-sandwich scaffold in larger LPMO10s. Despite these deletions, the enzyme is active on alpha- and beta-chitin, and the chitin-binding surface previously described for larger LPMOs is fully conserved. JdLPMO10A may represent a minimal scaffold needed to catalyse the powerful LPMO reaction.

Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra-modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin-type III (FnIII)-like domains, and a carbohydrate-binding module (CBM5), is a powerful chitin-active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin.

The AAA ATPase spastin is a microtubule-severing enzyme that plays important roles in various cellular events including axon regeneration. Herein, we found that the basal ATPase activity of spastin is negatively regulated by spastin concentration. By determining a spastin crystal structure, we demonstrate the necessity of intersubunit interactions between spastin AAA domains. Neutralization of the positive charges in the microtubule-binding domain (MTBD) of spastin dramatically decreases the ATPase activity at low concentration, although the ATP-hydrolyzing potential is not affected. These results demonstrate that, in addition to the AAA domain, the MTBD region of spastin is also involved in regulating ATPase activity, making interactions between spastin protomers more complicated than expected.

Immediate-early response 5 (IER5) is a growth factor-inducible protein with homology to the N-terminus of IER2. Deletion analysis shows that a large region of IER5, including the N-terminal region, is involved in cell growth and stress resistance. The N-terminal region mediates IER5 oligomerization and binding to the B55 regulatory subunit of protein phosphatase 2A (PP2A). IER5 physically interacts with the PP2A target proteins ribosomal protein S6 kinase (S6K) and heat shock factor 1 (HSF1), and the interactions are essential for the reduced phosphorylation of S6K and HSF1. Our data indicate that oligomeric IER5 regulates PP2A activity and cell growth.

The membrane protein transhydrogenase in animal mitochondria and bacteria couples reduction of NADP+ by NADH to proton translocation. Recent X-ray data on Thermus thermophilus transhydrogenase indicate a significant difference in the orientations of the two dIII components of the enzyme dimer (Leung et al., 2015). The character of the orientation change, and a review of information on the kinetics and thermodynamics of transhydrogenase, indicate that dIII swivelling might assist in the control of proton gating by the redox state of bound NADP+/NADPH during enzyme turnover.

The HOPS multisubunit tethering factor (MTC) is a macromolecular protein complex composed of six different subunits. It is one of the key components in the perception and subsequent fusion of multivesicular bodies and vacuoles. Electron microscopy studies indicate structural flexibility of the purified HOPS complex. Inducing higher rigidity into HOPS by biochemically modifying the complex declines the potential to mediate SNARE-driven membrane fusion. Thus, we propose that integral flexibility seems to be not only a feature, but of essential need for the function of HOPS. This review focuses on the general features of membrane tethering and fusion. For this purpose, we compare the structure and mode of action of different tethering factors to highlight their common central features and mechanisms.

In this study, we investigated the mechanism of O2 tolerance of Klebsiella oxytoca HP1 H2-evolving hydrogenase 3 (KHyd3) by mutational analysis and three-dimensional structure modeling. Results revealed that certain surface amino acid residues of KHyd3 large subunit, in particular those at the outer entrance of the gas channel, have a visible effect on its oxygen tolerance. Additionally, solution pH, immobilization and O2 partial pressure also affect KHyd3 O2-tolerance to some extent. We propose that the extent of KHyd3 O2-tolerance is determined by a balance between the rate of O2 access to the active center through gas channels and the deoxidation rate of the oxidized active center. Based on our findings, two higher O2-tolerant KHyd3 mutations G300E and G300M were developed.

PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B', B'' and B'''/striatin. Here, we found that a distinct PP2A complex regulates NF-κB signalling by dephosphorylation of IKKβ, IκBα and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB'''C trimer dephosphorylate IKKβ, IκBα and RelA, whereas the ABC trimer dephosphorylates IκBα but not IKKβ and RelA in cells. In contrast, AB'C and AB''C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF-κB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF-κB- and IKKβ-mediated cellular responses.

In archaea the A

We investigated H+ and Na+ transport by complex I from Escherichia coli devoid of the NuoL subunit, which is probably part of the ion translocating machinery. We observed that complex I devoid of the NuoL subunit still translocates H+, although to a smaller extension than the complete version of complex I, but does not transport Na+. Our results unequivocally reinforce the observation that E. coli complex I transports Na+ in the opposite direction to that of the H+ and show that NuoL subunit is involved in the translocation of both ions by complex I.

During leaf senescence in Arabidopsis, STAYGREEN 1 (SGR1) and SGR2 regulate chlorophyll degradation positively and negatively, respectively. SGR-LIKE (SGRL) is also expressed in pre-senescing leaves, but its function remains largely unknown. Here we show that under abiotic stress, Arabidopsis plants overexpressing SGRL exhibit early leaf yellowing and sgrl-1 mutants exhibit persistent green color of leaves. Under salt stress, SGR1 and SGRL act synergistically for rapid Chl degradation prior to senescence. Furthermore, SGRL forms homo- and heterodimers with SGR1 and SGR2 in vivo, and interacts with LHCII and chlorophyll catabolic enzymes. The role of SGRL under abiotic stress is discussed.

Highlights • Crystal structure of cohesin subunit Scc3/SA, showing irregular HEAT-like repeats. • Scc3 C-terminal domain binds Scc1, cohesin’s kleisin. • Scc1’s Scc3 binding region mapped. • Scc3 turns over in G2/M while maintaining cohesin’s association with chromosomes. • Scc3 promotes de-acetylation of Smc3 upon Scc1 cleavage., Sister chromatid cohesion involves entrapment of sister DNAs by a cohesin ring created through association of a kleisin subunit (Scc1) with ATPase heads of Smc1/Smc3 heterodimers. Cohesin’s association with chromatin involves subunits recruited by Scc1: Wapl, Pds5, and Scc3/SA, in addition to Scc2/4 loading complex. Unlike Pds5, Wapl, and Scc2/4, Scc3s are encoded by all eukaryotic genomes. Here, a crystal structure of Scc3 reveals a hook-shaped protein composed of tandem α helices. Its N-terminal domain contains a conserved and essential surface (CES) present even in organisms lacking Pds5, Wapl, and Scc2/4, while its C-terminal domain binds a section of the kleisin Scc1. Scc3 turns over in G2/M while maintaining cohesin’s association with chromosomes and it promotes de-acetylation of Smc3 upon Scc1 cleavage.

Post-translational modification of forkhead family transcription factor, FoxO1, is an important regulatory mode for its diverse activities. FoxO1 is acetylated by HAT coactivators and its transcriptional activity is decreased via reduced DNA binding affinity. Here, we report that SET/TAF-Iβ inhibited p300-mediated FoxO1 acetylation in an INHAT domain-dependent manner. SET/TAF-Iβ interacted with FoxO1 and activated transcription of FoxO1 target gene, p21. Moreover, SET/TAF-Iβ inhibited acetylation of FoxO1 and increased p21 transcription induced by oxidative stress. Our results suggest that SET/TAF-Iβ inhibits FoxO1 acetylation and activates its transcriptional activity toward p21.

Mammalian complex I is composed of fourteen highly conserved core subunits and additional thirty subunits acquired in the course of evolution. At present, the function of the majority of these supernumerary subunits is poorly understood. In this work, we have studied NDUFA3, NDUFA5 and NDUFA12 supernumerary subunits to gain insight into their role in CI activity and biogenesis. Using human cell lines in which the expression of these subunits was knocked down with miRNAs, we showed that they are necessary for the formation of a functional holoenzyme. Analysis of the assembly intermediates in mitochondria depleted for these subunits further suggested that they are required for assembly and/or stability of the electron transferring Q module in the peripheral arm of the CI.

The activity of the respiratory enzyme fumarate reductase (FRD) is dependent on the covalent attachment of the redox cofactor flavin adenine dinucleotide (FAD). We demonstrate that the FAD assembly factor SdhE, which flavinylates and activates the respiratory enzyme succinate dehydrogenase (SDH), is also required for the complete activation and flavinylation of FRD. SdhE interacted with, and flavinylated, the flavoprotein subunit FrdA, whilst mutations in a conserved RGxxE motif impaired the complete flavinylation and activation of FRD. These results are of widespread relevance because SDH and FRD play an important role in cellular energetics and are required for virulence in many important bacterial pathogens.

Mammalian SWI/SNF or BAF chromatin-remodeling complexes are polymorphic assemblies of homologous subunit families that remodel nucleosomes. BAF57 is a subunit of the BAF complexes; it is encoded only in higher eukaryotes and is present in all mammalian assemblies. Its main structural feature is a high-mobility group domain, the DNA-binding properties of which suggest that BAF57 may play topological roles as the BAF complex enters or exits the nucleosome. BAF57 displays specific interactions with a number of proteins outside the BAF complex. Through these interactions, it can accomplish specific functions. In the embryo, BAF57 is responsible for the silencing of the CD4 gene during T-cell differentiation, and during the repression of neuronal genes in non-neuronal cells, BAF57 interacts with the transcriptional corepressor, Co-REST, and facilitates repression. Extensive work has demonstrated a specific role of BAF57 in regulating the interactions between BAF and nuclear hormone receptors. Despite its involvement in oncogenic pathways, new generation sequencing studies do not support a prominent role for BAF57 in the initiation of cancer. On the other hand, evidence has emerged to support a role for BAF57 as a metastasis factor, a prognosis marker and a therapeutic target. In humans, BAF57 is associated with disease, as mutations in this gene predispose to important congenital disorders, including menigioma disease or the Coffin-Siris syndrome. In this article, we present an exhaustive analysis of the BAF57 molecular and biochemical properties, cellular functions, loss-of-function phenotypes in living organisms and pathological manifestations in cases of human mutations.

N-myristoylation is a lipid modification of many signaling proteins in which myristate is added to an N-terminal glycine residue. Here we show that PP2C74, a putative myristoylated 2C-type protein phosphatase (PP2C) in Arabidopsis, is transcribed in various tissues and has protein phosphatase activity. GFP-fused PP2C74 localized to the plasma membrane, but not when a glycine residue at position 2, which is the putative myristoylation site, was substituted with an alanine residue. Yeast two-hybrid analysis and GST pull-down analysis showed that PP2C74 interacts with AKIN10, the catalytic α subunit of the SnRK1 protein kinase complex, the β subunits of which are known targets of myristoylation.Structured summary of protein interactionsAKIN10 physically interacts with PP2C74 by two hybrid (View interaction)AKIN10 physically interacts with PP2C74 by pull down (View interaction)

The short pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) constitute a group of innate immune receptors that trigger immune activation by detecting molecules of the microbial cell wall. Here, we examined the evolution of short pentraxins in Murinae lineages. By molecular evolutionary analysis, CRP and SAP have been experiencing rapid diversification, driven by adaptive selection. Further, our protein modeling demonstrates that adaptively selected amino acids lie in the ligand-binding region and contact region between subunits. Our findings suggest that rapid diversification of these regions could contribute to the determinants of recognizing specificity and the interaction between subunits.

Although the exact etiology and pathogenesis of Alzheimer's disease (AD) are still unclear, amyloid-β (Aβ) generated by the proteolytic processing of amyloid-β precursor protein (APP) aggregate to form toxic amyloid species. Kinesin-1 is the first identified ATP-dependent axonal transport motor protein that has been proven to affect Aβ generation and deposition. In this paper, we applied dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) to investigate the direct interaction of Aβ with kinesin-1 at the single-molecule fluorescence level in vitro. The results showed that two kinds of enhanced green fluorescent protein (EGFP)-tagged kinesin light-chain subunits of kinesin-1(KLCs), KLC-E and E-KLC inhibited the aggregation of Aβ over a period of time, providing additional insight into the mechanism of axonal transport deficits in AD.

The MCM (mini-chromosome maintenance) complex is the core of the eukaryotic replicative helicase and comprises six proteins, Mcm2–Mcm7. In humans, a variant form of the complex has Mcm2 replaced by the MCM–BP protein. Recent results suggest that a similar complex exists in fission yeast with an essential role in DNA replication and cell cycle progression. Here, we describe the purification and subunit composition of the fission yeast MCMMcb1 complex. Using newly generated temperature-sensitive alleles, we show that loss of MCMMcb1 function leads to accumulation of DNA damage, checkpoint activation and cell cycle arrest, and provide evidence for a role for MCMMcb1 in meiosis. Structured summary of protein interactions Mcb1 physically interacts with Mcm4 by pull down ( View interaction ) Mcb1 physically interacts with Mcm5 by pull down ( View interaction ) Mcb1 physically interacts with Mcm6 by pull down ( View interaction ) Mcm4 and Mcb1 physically interact by bimolecular fluorescence complementation ( View interaction ) Mcb1 physically interacts with Mcm3 , Mcm4 , Mcm5 , Mcm6 and Mcm7 by tandem affinity purification ( View interaction ) Mcb1 physically interacts with Mcm7 by pull down ( View interaction )

Protein phosphatase-2A (PP2A) activity, which is compromised in Alzheimer disease brain, is regulated by two endogenous inhibitors, one of them being I2PP2A, a 277 amino acid long protein also known as SET. Here we report that both the amino terminal fragment (I2NTF; aa 1–175) and the carboxy terminal fragment (I2CTF; aa 176–277) of I2PP2A inhibit PP2A by binding to its catalytic subunit PP2Ac and cause hyperphosphorylation of tau. The C-terminal acidic region in I2CTF and Val 92 in I2NTF are essential for their association with PP2Ac and inhibition of the phosphatase activity. Structured summary of protein interactions I2PP2A physically interacts with PP2A-C by anti tag coimmunoprecipitation (view interaction 1 , 2 ) I2PP2A physically interacts with PP2A-C by pull down (view interaction 1 , 2 ) PP2A-A physically interacts with PP2A-B and PP2A-C by pull down (view interaction)

Archaea-specific D-family DNA polymerase forms a heterotetramer consisting of two large polymerase subunits and two small exonuclease subunits. The N-terminal (1–300) domain structure of the large subunit was determined by X-ray crystallography, although ∼50 N-terminal residues were disordered. The determined structure consists of nine alpha helices and three beta strands. We also identified the DNA-binding ability of the domain by SPR measurement. The N-terminal (1–100) region plays crucial roles in the folding of the large subunit dimer by connecting the ∼50 N-terminal residues with their own catalytic region (792–1163).Structured summaryDP2 binds to DP2 by molecular sieving (View interaction)DP2 binds to DP2 by fluorescence technology (View interaction)DP2 binds to DP2 by circular dichroism (View interaction)

The chlorella virus-encoded Kcv can form a homo-tetrameric potassium channel in lipid membranes. This miniature peptide can be synthesized in vitro, and the tetramer purified from the SDS–polyacrylamide gel retains the K+ channel functionality. Combining this capability with the mass-tagging method, we propose a simple, straightforward approach that can generically manipulate individual subunits in the tetramer, thereby enabling the detection of contribution from individual subunits to the channel functions. Using this approach, we showed that the structural change in the selectivity filter from only one subunit is sufficient to cause permanent channel inactivation (“all-or-none” mechanism), whereas the mutation near the extracellular entrance additively modifies the ion permeation with the number of mutant subunits in the tetramer (“additive” mechanism).

NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [3H]benzophenone-asimicin ([3H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [3H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS–PAGE) of [3H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding.

The universal enzymatic cofactor vitamin B6 can be synthesized as pyridoxal 5-phosphate (PLP) by the glutamine amidotransferase Pdx1. We show that Saccharomyces cerevisiae Pdx1 is hexameric by analytical ultracentrifugation and by crystallographic 3D structure determination. Bacterial homologues were previously reported to exist in hexamer:dodecamer equilibrium. A small sequence insertion found in yeast Pdx1 elevates the dodecamer dissociation constant when introduced into Bacillus subtilis Pdx1. Further, we demonstrate that the yeast Pdx1 C-terminus contacts an adjacent subunit, and deletion of this segment decreases enzymatic activity 3.5-fold, suggesting a role in catalysis.Structured summaryMINT-7147859: PDX1 (uniprotkb:P16451) and PDX1 (uniprotkb:P16451) bind (MI:0407) by cosedimentation in solution (MI:0028)MINT-7147899: PDX1 (uniprotkb:P37528) and PDX1 (uniprotkb:P37528) bind (MI:0407) by cosedimentation in solution (MI:0028)

Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits. To examine this possibility, LptB was expressed with LptF and LptG. We report here that both LptF and LptG formed a complex with LptB. Furthermore, an inner membrane protein, LptC, which had been implicated in lipopolysaccharide transport, was also included in this complex.Structured summaryMINT-7137021: lptb (uniprotkb:P0A9V1) physically interacts (MI:0914) with lptc (uniprotkb:P0ADV9), lptg (uniprotkb:P0ADC6) and lptf (uniprotkb:P0AF98) by pull down (MI:0096)MINT-7137160: lptb (uniprotkb:P0A9V1) physically interacts (MI:0914) with lptf (uniprotkb:P0AF98) and lptg (uniprotkb:P0ADC6) by pull down (MI:0096)

eIF3f is a subunit of eukaryotic initiation factor 3 (eIF3). We previously showed that eIF3f is phosphorylated by cyclin dependent kinase 11 (CDK11p46) which is an important effector in apoptosis. Here, we identified a second eIF3f phosphorylation site (Thr119) by CDK11p46 during apoptosis. We demonstrated that eIF3f is directly phosphorylated by CDK11p46 in vivo. Phosphorylation of eIF3f plays an important role in regulating its function in translation and apoptosis. Phosphorylation of eIF3f enhances the association of eIF3f with the core eIF3 subunits during apoptosis. Our data suggested that eIF3f may inhibit translation by increasing the binding to the eIF3 complex during apoptosis.Structured summaryMINT-6948874: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948891: EIF3b (uniprotkb:P55884) physically interacts (MI:0218) with EIF3c (uniprotkb:Q99613), EIF3a (uniprotkb:Q14152) and EIF3f (uniprotkb:O00303) by anti bait coimmunoprecipitation (MI:0006)MINT-6948836, MINT-6948849, MINT-6948862: CDK11p46 (uniprotkb:P21127) phosphorylates (MI:0217) EIF3f (uniprotkb:O00303) by protein kinase assay (MI:0424)

Escherichia coli RNase P is a ribonucleoprotein composed of a large RNA subunit (M1 RNA) and a small protein subunit (C5 protein). We examined if C5 protein plays a role in maintaining metabolic stability of M1 RNA. The sequestration of C5 protein available for M1 RNA binding reduced M1 RNA stability in vivo, and its reduced stability was recovered via overexpression of C5 protein. In addition, M1 RNA was rapidly degraded in a temperature-sensitive C5 protein mutant strain at non-permissive temperatures. Collectively, our results demonstrate that the C5 protein metabolically stabilizes M1 RNA in the cell.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, reacts with three different Sox proteins. Its active site Cys110Y is on the carboxy-terminus of the SoxY subunit. SoxYZ “as isolated” consisted mainly of the catalytically inactive SoxY-Y(Z)2 heterotetramer linked by a Cys110Y-Cys110Y interprotein disulfide. Sulfide activated SoxYZ “as isolated” 456-fold, reduced the disulfide, and yielded an active SoxYZ heterodimer. The reductant tris(2-carboxyethyl)phosphine (TCEP) inactivated SoxYZ. This form was not re-activated by sulfide, which identified it as a different inactive form. In analytical gel filtration, the elution of “TCEP-treated” SoxYZ was retarded compared to active SoxYZ, indicating a conformational change. The possible enzymes involved in the re-activation of each inactive form of SoxYZ are discussed.

Halomonas nucleoside diphosphate kinase (HaNDK) forms a dimeric assembly and Pseudomonas NDK (PaNDK) forms a tetrameric assembly. The mutation of Glu134 to Ala in HaNDK resulted in the conversion of the native dimeric structure to the tetramer assembly. Conversely, the mutation of Ala134 to Glu in PaNDK lead to the conversion from the tetramer to the dimer assembly, indicating that a single amino acid substitution at position 134 results in an alteration of the oligomeric structure of NDK. By modeling the structure of HaNDK and PaNDK based on the crystal structure of Myxococcus NDK, we showed that Glu134 exerts sufficient repulsive forces to disrupt the dimer–dimer interaction and prevent the formation of the tetramer.

A novel ferritin cDNA, SferH-5, has been cloned from 7-day-old soybean seedlings. Putative SferH-5 has 96% identity with SferH-1 reported previously. All the five amino acid variants distributed in the mature region are not involved in highly conserved residues associated with ferroxidase activity center. We speculate that SferH-5 encodes a novel 26.5-kDa subunit of soybean seed ferritin, which is designated H-5 in this study. Recombinant H-5 was able to assemble, together with co-expressed H-2, as a functional soybean seed ferritin-like complex, H-5/H-2. Our data reveal the potential heterogeneity of the 26.5-kDa subunit of soybean seed ferritin.

The inhibition of hepatic glycogen-associated protein phosphatase-1 (PP1-G(L)) by glycogen phosphorylase a prevents the dephosphorylation and activation of glycogen synthase, suppressing glycogen synthesis when glycogenolysis is activated. Here, we show that a peptide ((280)LGPYY(284)) comprising the last five amino acids of G(L) retains high-affinity interaction with phosphorylase a and that the two tyrosines play crucial roles. Tyr284 deletion abolishes binding of phosphorylase a to G(L) and replacement by phenylalanine is insufficient to restore high-affinity binding. We show that a phosphorylase inhibitor blocks the interaction of phosphorylase a with the G(L) C-terminus, suggesting that the latter interaction could be targeted to develop an anti-diabetic drug.

Here we show that the cell-surface expression of the alpha subunit of H(+)-ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H(+)-ATP synthase or antibodies against alpha and beta subunits of H(+)-ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A-I, which has been shown to bind to the ectopic beta-chain of H(+)-ATP synthase and inhibit the activity of cell-surface H(+)-ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell-surface H(+)-ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.

Based on the characterization of ribosome precursor particles and associated trans-acting factors, a biogenesis pathway for the 40S and 60S subunits has emerged. After nuclear synthesis and assembly steps, pre-ribosomal subunits are exported through the nuclear pore complex in a Crm1- and RanGTP-dependent manner. Subsequent cytoplasmic biogenesis steps of pre-60S particles include the facilitated release of several non-ribosomal proteins, yielding fully functional 60S subunits. Cytoplasmic maturation of 40S subunit precursors includes rRNA dimethylation and pre-rRNA cleavage, allowing 40S subunits to achieve translation competence. We review current knowledge of nuclear export and cytoplasmic maturation of ribosomal subunits.

In Drosophila, developing germline cysts in testis are enveloped by two somatic cyst cells essential for germline development and male reproduction. The cyst cells continue development along with the germline. However, the mechanisms of somatic gene expression in testes are poorly understood. We report transcriptional up-regulation of the Ku heterodimer in cyst cells. The initial up-regulation is independent of germline, and transcription is further augmented during spermatogenesis. Abundance of Ku in the cyst cell cytoplasm suggests the role for Ku subunits in the regulation of sperm individualization.

The multi-enzyme complex glycine decarboxylase is important for one-carbon metabolism, essential for the photorespiratory glycolate cycle of plants, and comprises four different polypeptides, P-, H-, T-, and L-protein. We report on the production and properties of recombinant P-protein from the cyanobacterium Synechocystis and also describe features of recombinant H-protein from the same organism. The P-protein shows enzymatic activity with lipoylated H-protein and very low activity with H-apoprotein or lipoate as artificial cofactors. Its affinity towards glycine is unaffected by the presence and nature of the methyleneamine acceptor molecule. The cyanobacterial H-protein apparently forms stable dimers.

We have mapped the sites of ischemia/reperfusion-induced phosphorylation of cytochrome c oxidase (CcO) subunits in rabbit hearts by using a combination of Blue Native gel/Tricine gel electrophoresis and nano-LC–MS/MS approaches. We used precursor ion scanning combined with neutral loss scanning and found that mature CcO subunit I was phosphorylated at tandem Ser115/Ser116 positions, subunit IVi1 at Thr52 and subunit Vb at Ser40. These sites are highly conserved in mammalian species. Molecular modeling suggests that phosphorylation sites of subunit I face the inter membrane space while those of subunits IVi1 and Vb face the matrix side.

P4-ATPases, or flippases, translocate phospholipids between the two leaflets of eukaryotic biological membranes. They are essential to the physiologically crucial phospholipid asymmetry and involved in severe diseases, but their molecular structure and mechanism are still unresolved. Here, we show that in an extensive mutational alanine screening of the mammalian flippase ATP8A2 catalytic subunit, five mutations stand out by leading to reduced glycosylation of the accessory subunit CDC50A. These mutations may disturb the interaction between the subunits.

A series of luciferase reporter constructs was prepared from a 1035-bp fragment of mouse genomic DNA flanking the 5′-coding sequence for the SPTLC2 subunit of serine palmitoyltransferase, the initial enzyme of de novo sphingolipid biosynthesis. The full-length DNA fragment promoted strong reporter gene expression in NIH3T3 cells while deletion and site-directed mutagenesis indicated that the proximal 335bp contain initiator and downstream promoter elements, two proximal GC boxes that appear to stimulate transcription in a cooperative manner, and several additional elements whose activity cannot be accounted for by known factor binding sites. These findings provide insight into the control mechanisms for transcription of mammalian SPTLC2.