The LYR protein subunit NB4M/NDUFA6 of mitochondrial complex I anchors an acyl carrier protein and is essential for catalytic activity.

Mitochondrial complex I is the largest and most complicated enzyme of the oxidative phosphorylation system. It comprises a number of so-called accessory subunits of largely unknown structure and function. Here we studied subunit NB4M [NDUFA6, LYR motif containing protein 6 (LYRM6)], a member of the LYRM family of proteins. Chromosomal deletion of the corresponding gene in the yeast Yarrowia lipolytica caused concomitant loss of the mitochondrial acyl carrier protein subunit ACPM1 from the enzyme complex and paralyzed ubiquinone reductase activity. Exchanging the LYR motif and an associated conserved phenylalanine by alanines in subunit NB4M also abolished the activity and binding of subunit ACPM1. We show, by single-particle electron microscopy and structural modeling, that subunits NB4M and ACPM1 form a subdomain that protrudes from the peripheral arm in the vicinity of central subunit domains known to be involved in controlling the catalytic activity of complex I.

Functional dissection of the proton pumping modules of mitochondrial complex I.

Mitochondrial complex I, the largest and most complicated proton pump of the respiratory chain, links the electron transfer from NADH to ubiquinone to the pumping of four protons from the matrix into the intermembrane space. In humans, defects in complex I are involved in a wide range of degenerative disorders. Recent progress in the X-ray structural analysis of prokaryotic and eukaryotic complex I confirmed that the redox reactions are confined entirely to the hydrophilic peripheral arm of the L-shaped molecule and take place at a remarkable distance from the membrane domain. While this clearly implies that the proton pumping within the membrane arm of complex I is driven indirectly via long-range conformational coupling, the molecular mechanism and the number, identity, and localization of the pump-sites remains unclear. Here, we report that upon deletion of the gene for a small accessory subunit of the Yarrowia complex I, a stable subcomplex (nb8mΔ) is formed that lacks the distal part of the membrane domain as revealed by single particle analysis. The analysis of the subunit composition of holo and subcomplex by three complementary proteomic approaches revealed that two (ND4 and ND5) of the three subunits with homology to bacterial Mrp-type Na(+)/H(+) antiporters that have been discussed as prime candidates for harbouring the proton pumps were missing in nb8mΔ. Nevertheless, nb8mΔ still pumps protons at half the stoichiometry of the complete enzyme. Our results provide evidence that the membrane arm of complex I harbours two functionally distinct pump modules that are connected in series by the long helical transmission element recently identified by X-ray structural analysis.

A scaffold of accessory subunits links the peripheral arm and the distal proton-pumping module of mitochondrial complex I.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is a very large membrane protein complex with a central function in energy metabolism. Complex I from the aerobic yeast Yarrowia lipolytica comprises 14 central subunits that harbour the bioenergetic core functions and at least 28 accessory subunits. Despite progress in structure determination, the position of individual accessory subunits in the enzyme complex remains largely unknown. Proteomic analysis of subcomplex Iδ revealed that it lacked eleven subunits, including the central subunits ND1 and ND3 forming the interface between the peripheral and the membrane arm in bacterial complex I. This unexpected observation provided insight into the structural organization of the connection between the two major parts of mitochondrial complex I. Combining recent structural information, biochemical evidence on the assignment of individual subunits to the subdomains of complex I and sequence-based predictions for the targeting of subunits to different mitochondrial compartments, we derived a model for the arrangement of the subunits in the membrane arm of mitochondrial complex I.

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L.

Here we study ATP synthase from human rho0 (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF1 inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF1 still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in rho0 cells and not to the dimeric form. This supports previous suggestions that IF1 plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.

The serine/threonine kinase Stk and the phosphatase Stp regulate cell wall synthesis in Staphylococcus aureus.

The cell wall synthesis pathway producing peptidoglycan is a highly coordinated and tightly regulated process. Although the major components of bacterial cell walls have been known for decades, the complex regulatory network controlling peptidoglycan synthesis and many details of the cell division machinery are not well understood. The eukaryotic-like serine/threonine kinase Stk and the cognate phosphatase Stp play an important role in cell wall biosynthesis and drug resistance in S. aureus. We show that stp deletion has a pronounced impact on cell wall synthesis. Deletion of stp leads to a thicker cell wall and decreases susceptibility to lysostaphin. Stationary phase Δstp cells accumulate peptidoglycan precursors and incorporate higher amounts of incomplete muropeptides with non-glycine, monoglycine and monoalanine interpeptide bridges into the cell wall. In line with this cell wall phenotype, we demonstrate that the lipid II:glycine glycyltransferase FemX can be phosphorylated by the Ser/Thr kinase Stk in vitro. Mass spectrometric analyses identify Thr32, Thr36 and Ser415 as phosphoacceptors. The cognate phosphatase Stp dephosphorylates these phosphorylation sites. Moreover, Stk interacts with FemA and FemB, but is unable to phosphorylate them. Our data indicate that Stk and Stp modulate cell wall synthesis and cell division at several levels.

Rab7-a novel redox target that modulates inflammatory pain processing.

Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.

Generator-specific targets of mitochondrial reactive oxygen species.

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for "signaling" and "damaging" ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc1 complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H2O2) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.

Inactivation of tristetraprolin in chronic hypoxia provokes the expression of cathepsin B.

Macrophages play important roles in many diseases and are frequently found in hypoxic areas. A chronic hypoxic microenvironment alters global cellular protein expression, but molecular details remain poorly understood. Although hypoxia-inducible factor (HIF) is an established transcription factor allowing adaption to acute hypoxia, responses to chronic hypoxia are more complex. Based on a two-dimensional differential gel electrophoresis (2D-DIGE) approach, we aimed to identify proteins that are exclusively expressed under chronic but not acute hypoxia (1% O2). One of the identified proteins was cathepsin B (CTSB), and a knockdown of either HIF-1α or -2α in primary human macrophages pointed to an HIF-2α dependency. Although chromatin immunoprecipitation (ChIP) experiments confirmed HIF-2 binding to a CTSB enhancer in acute hypoxia, an increase of CTSB mRNA was evident only under chronic hypoxia. Along those lines, CTSB mRNA stability increased at 48 h but not at 8 h of hypoxia. However, RNA stability at 8 h of hypoxia was enhanced by a knockdown of tristetraprolin (TTP). Inactivation of TTP under prolonged hypoxia was facilitated by c-Jun N-terminal kinase (JNK), and inhibition of this kinase lowered CTSB mRNA levels and stability. We postulate a TTP-dependent mechanism to explain delayed expression of CTSB under chronic hypoxia.

Age-related changes in the mitochondrial proteome of the fungus Podospora anserina analyzed by 2D-DIGE and LC-MS/MS.

Many questions concerning the molecular processes during biological aging remain unanswered. Since mitochondria are central players in aging, we applied quantitative two-dimensional difference gel electrophoresis (2D-DIGE) coupled to protein identification by mass spectrometry to study the age-dependent changes in the mitochondrial proteome of the fungus Podospora anserina - a well-established aging model. 67 gel spots exhibited significant, but remarkably moderate intensity changes. While typically the observed changes in protein abundance occurred progressively with age, for several proteins a pronounced change was observed at late age, sometimes inverting the trend observed at younger age. The identified proteins were assigned to a wide range of metabolic pathways including several implicated previously in biological aging. An overall decrease for subunits of complexes I and V of oxidative phosphorylation was confirmed by Western blot analysis and blue-native electrophoresis. Changes in several groups of proteins suggested a general increase in protein biosynthesis possibly reflecting a compensatory mechanism for increased quality control-related protein degradation at later age. Age-related augmentation in abundance of proteins involved in biosynthesis, folding, and protein degradation pathways sustain these observations. Furthermore, a significant decrease of two enzymes involved in the degradation of γ-aminobutyrate (GABA) supported its previously suggested involvement in biological aging. BIOLOGICAL SIGNIFICANCE: We have followed the time course of changes in protein abundance during aging of the fungus P. anserina. The observed moderate but significant changes provide insight into the molecular adaptations to biological aging and highlight the metabolic pathways involved, thereby offering new leads for future research.

Protein S-nitrosylation and denitrosylation in the mouse spinal cord upon injury of the sciatic nerve.

Nitric oxide is a pain signaling molecule and exerts its influence through two primary pathways: by stimulation of soluble guanylylcyclase and by direct S-nitrosylation (SNO) of target proteins. We assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME). After nerve injury, SNO-DIGE revealed 30 proteins with increased and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 proteins. After sham surgery only 3 proteins were up-nitrosylated. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7days showed that SNO modifications of protein disulfide isomerase, glutathione synthase and peroxiredoxin-6 had returned to baseline within 7days whereas S-nitrosylation of mitochondrial aconitase 2 was further increased. The identified SNO modified proteins are involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. The data show that nitric oxide mediated S-nitrosylation contributes to the nerve injury-evoked pathology in nociceptive signaling pathways.

Complexome profiling identifies TMEM126B as a component of the mitochondrial complex I assembly complex.

Macromolecular complexes are essential players in numerous biological processes. They are often large, dynamic, and rather labile; approaches to study them are scarce. Covering masses up to ∼30 MDa, we separated the native complexome of rat heart mitochondria by blue-native and large-pore blue-native gel electrophoresis to analyze its constituents by mass spectrometry. Similarities in migration patterns allowed hierarchical clustering into interaction profiles representing a comprehensive analysis of soluble and membrane-bound complexes of an entire organelle. The power of this bottom-up approach was validated with well-characterized mitochondrial multiprotein complexes. TMEM126B was found to comigrate with known assembly factors of mitochondrial complex I, namely CIA30, Ecsit, and Acad9. We propose terming this complex mitochondrial complex I assembly (MCIA) complex. Furthermore, we demonstrate that TMEM126B is required for assembly of complex I. In summary, complexome profiling is a powerful and unbiased technique allowing the identification of previously overlooked components of large multiprotein complexes.

Intramembrane proteolysis of Mgm1 by the mitochondrial rhomboid protease is highly promiscuous regarding the sequence of the cleaved hydrophobic segment.

Rhomboids are a family of intramembrane serine proteases that are conserved in bacteria, archaea, and eukaryotes. They are required for numerous fundamental cellular functions such as quorum sensing, cell signaling, and mitochondrial dynamics. Mitochondrial rhomboids form an evolutionarily distinct class of rhomboids. It is largely unclear how their activity is controlled and which substrate determinants are responsible for recognition and cleavage. We investigated these requirements for the mitochondrial rhomboid protease Pcp1 and its substrate Mgm1. In contrast to several other rhomboid proteases, Pcp1 does not require helix-breaking amino acids in the cleaved hydrophobic region of Mgm1, termed 'rhomboid cleavage region' (RCR). Even transmembrane segments of inner membrane proteins that are normally not processed by Pcp1 become cleavable when put in place of the authentic RCR of Mgm1. We further show that mutational alterations of a highly negatively charged region located C-terminally to the RCR led to a strong processing defect. Moreover, we show that the determinants required for Mgm1 processing by mitochondrial rhomboid protease are conserved during evolution, as PARL (the human ortholog of Pcp1) showed similar substrate requirements. These results suggest a surprising promiscuity of the mitochondrial rhomboid protease regarding the sequence requirements of the cleaved hydrophobic segment. We propose a working hypothesis on how the mitochondrial rhomboid protease can, despite this promiscuity, achieve a high specificity in recognizing Mgm1. This hypothesis relates to the exceptional biogenesis pathway of Mgm1.