Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

<p>Abstract</p> <p>Background</p> <p>The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of <it>β</it>-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the <it>β</it>-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs.</p> <p>Results</p> <p>We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on <it>β</it>-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and <it>β</it>-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the <it>β</it>-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a <it>β</it>-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive.</p> <p>Conclusions</p> <p>Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are <it>β</it>-signal dependent, few MBOMP families remain undiscovered in any sequenced organism.</p

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F0F1 ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F0F1 ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F1 subunits, three to F0 subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F1 α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F0F1-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought

A Deubiquitylating Complex Required for Neosynthesis of a Yeast Mitochondrial ATP Synthase Subunit

The ubiquitin system is known to be involved in maintaining the integrity of mitochondria, but little is known about the role of deubiquitylating (DUB) enzymes in such functions. Budding yeast cells deleted for UBP13 and its close homolog UBP9 displayed a high incidence of petite colonies and slow respiratory growth at 37°C. Both Ubp9 and Ubp13 interacted directly with Duf1 (DUB-associated factor 1), a WD40 motif-containing protein. Duf1 activates the DUB activity of recombinant Ubp9 and Ubp13 in vitro and deletion of DUF1 resulted in the same respiratory phenotype as the deletion of both UBP9 and UBP13. We show that the mitochondrial defects of these mutants resulted from a strong decrease at 37°C in the de novo biosynthesis of Atp9, a membrane-bound component of ATP synthase encoded by mitochondrial DNA. The defect appears at the level of ATP9 mRNA translation, while its maturation remained unchanged in the mutants. This study describes a new role of the ubiquitin system in mitochondrial biogenesis

Mitochondrial ATP synthase: architecture, function and pathology

Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F1, situated in the mitochondrial matrix, and Fo, located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions

Substitutions of potentially phosphorylatable serine residues of Bax reveal how they may regulate its interaction with mitochondria.

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The cytosolic domain of human Tom22 modulates human Bax mitochondrial translocation and conformation in yeast

International audienceThe role of the mitochondrial protein receptor Tom22p in the interaction of pro-apoptotic protein Bax with yeast mitochondria was investigated. Co-immunoprecipitation assays showed that human Bax interacted with different TOM subunits, including Tom22p. Expression of the cytosolic receptor domain of human Tom22 increased Bax mitochondrial localization, but decreased the proportion of active Bax. BN-PAGE showed that the cytosolic domain of Tom22 interfered with the oligomerization of Bax. These data suggest that the interaction with the cytosolic domain of Tom22 helps Bax to acquire a conformation able to interact with the outer mitochondrial membrane.Structured summary of protein interactions:BAX and BAX physically interact with TOM22 by anti bait coimmunoprecipitation (View interaction)BAX physically interacts with TOM70 by anti bait coimmunoprecipitation (View interaction)BAX physically interacts with TOM22, TOM70 and TOM40 by anti bait coimmunoprecipitation (View interaction)BAX physically interacts with TOM22 by anti bait coimmunoprecipitation (View interaction)BAX and BAX bind by blue native page (View interaction) BAX physically interacts with TOM40 by anti bait coimmunoprecipitation (View interaction

PSI1 is responsible for the stearic acid enrichment that is characteristic of phosphatidylinositol in yeast

In yeast, both phosphatidylinositol and phosphatidylserine are synthesized from cytidine diphosphate-diacylglycerol. Because, as in other eukaryotes, phosphatidylinositol contains more saturated fatty acids than phosphatidylserine (and other phospholipids), it has been hypothesized that either phosphatidylinositol is synthesized from distinct cytidine diphosphate-diacylglycerol molecules, or that, after its synthesis, it is modified by a hypothetical acyltransferase that incorporates saturated fatty acid into neo-synthesized molecules of phosphatidylinositol. We used database search methods to identify an acyltransferase that could catalyze such an activity. Among the various proteins that we studied, we found that Psi1p (phosphatidylinositol stearoyl incorporating 1 protein) is required for the incorporation of stearate into phosphatidylinositol because GC and MS analyses of psi1Delta lipids revealed an almost complete disappearance of stearic (but not of palmitic acid) at the sn-1 position of this phospholipid. Moreover, it was found that, whereas glycerol 3-phosphate, lysophosphatidic acid and 1-acyl lysophosphatidylinositol acyltransferase activities were similar in microsomal membranes isolated from wild-type and psi1Delta cells, microsomal membranes isolated from psi1Delta cells are devoid of the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity that is present in microsomal membranes isolated from wild-type cells. Moreover, after the expression of PSI1 in transgenic psi1Delta cells, the sn-2-acyl-1-lysolysophosphatidylinositol acyltransferase activity was recovered, and was accompanied by a strong increase in the stearic acid content of lysophosphatidylinositol. As previously suggested for phosphatidylinositol from animal cells (which contains almost exclusively stearic acid as the saturated fatty acid), the results obtained in the present study demonstrate that the existence of phosphatidylinositol species containing stearic acid in yeast results from a remodeling of neo-synthesized molecules of phosphatidylinositol