The role of the Tim8p–Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p–Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p–Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p–Tim13p complex is not dependent on zinc, and the purified Tim8p–Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains

Tim54p connects inner membrane assembly and proteolytic pathways in the mitochondrion

Tim54p, a component of the inner membrane TIM22 complex, does not directly mediate the import of inner membrane substrates but is required for assembly/stability of the 300-kD TIM22 complex. In addition, Δtim54 yeast exhibit a petite-negative phenotype (also observed in yeast harboring mutations in the F1Fo ATPase, the ADP/ATP carrier, mitochondrial morphology components, or the i–AAA protease, Yme1p). Interestingly, other import mutants in our strain background are not petite-negative. We report that Tim54p is not involved in maintenance of mitochondrial DNA or mitochondrial morphology. Rather, Tim54p mediates assembly of an active Yme1p complex, after Yme1p is imported via the TIM23 pathway. Defective Yme1p assembly is likely the major contributing factor for the petite-negativity in strains lacking functional Tim54p. Thus, Tim54p has two independent functions: scaffolding/stability for the TIM22 membrane complex and assembly of Yme1p into a proteolytically active complex. As such, Tim54p links protein import, assembly, and turnover pathways in the mitochondrion

Evaluation of brain mitochondrial glutamate and alpha-ketoglutarate transport under physiologic conditions

Some models of brain energy metabolism used to interpret in vivo (13)C nuclear magnetic resonance spectroscopic data assume that intramitochondrial alpha-ketoglutarate is in rapid isotopic equilibrium with total brain glutamate, most of which is cytosolic. If so, the kinetics of changes in (13)C-glutamate can be used to predict citric acid cycle flux. For this to be a valid assumption, the brain mitochondrial transporters of glutamate and alpha-ketoglutarate must operate under physiologic conditions at rates much faster than that of the citric acid cycle. To test the assumption, we incubated brain mitochondria under physiologic conditions, metabolizing both pyruvate and glutamate and measured rates of glutamate, aspartate, and alpha-ketoglutarate transport. Under the conditions employed (66% of maximal O(2) consumption), the rate of synthesis of intramitochondrial alpha-ketoglutarate was 142 nmol/min.mg and the combined initial rate of alpha-ketoglutarate plus glutamate efflux from the mitochondria was 95 nmol/min.mg. It thus seems that much of the alpha-ketoglutarate synthesized within the mitochondria proceeds around the citric acid cycle without equilibrating with cytosolic glutamate. Unless the two pools are in such rapid exchange that they maintain the same percent (13)C enrichment at all points, (13)C enrichment of glutamate alone cannot be used to determine tricarboxylic acid cycle flux. The alpha-ketoglutarate pool is far smaller than the glutamate pool and will therefore approach steady state faster than will glutamate at the metabolite transport rates measured

Physical interaction and functional studies of human SCO1, a mitochondrial metallochaperone

Cytochrome c oxidase (COX) is a multimeric protein complex embedded in the inner mitochondrial membrane that contributes to the electrochemical potential ultimately required for adenosine triphosphate (ATP) synthesis. Synthesis of Cytochrome c Oxidase 1 (SCO1) and SCO2 are two of many accessory proteins that facilitate the assembly of individual COX structural subunits into a functional holoenzyme complex. SCO1 and SCO2 also function to regulate cellular copper homeostasis. Both of these functions require that SCO proteins collaborate with several interacting partners. ¬With few exceptions, however, their protein partners have yet to be identified and, as a consequence, we lack mechanistic insight into SCO protein function. To address this gap in our knowledge, I used physical methods to identify interacting partners of SCO1. Physical interactions between potential interacting partners of endogenous SCO1 or overexpressed SCO1-FLAG were stabilized with a chemical crosslinker, and the protein complexes were purified using the appropriate primary antibody. Mass spectrometric analysis of the eluates provided two lists of potential interacting partners that were subsequently filtered by gene ontology function and mitochondrial localization. The final list was comprised of 85 proteins, which were rank ordered based on total peptide counts per protein. Three of these candidate interacting partners were then further characterized; COX20, tricarboxylate transport protein (SLC25A1) and mitochondrial 2-oxoglutarate/malate carrier (SLC25A11). I first confirmed the authenticity of the observed interactions by conducting the reciprocal co-immunoprecipitations in the absence of a chemical crosslinker. I then investigated the effect of transient knockdown of each protein on the abundance of COX II, a direct proxy of total COX content. Knockdown of COX20 and SLC25A1 reduced COX II levels in SCO1 and SCO2 patient fibroblasts, but did not affect COX II abundance in control cells. SLC25A11 knockdown lowered COX II abundance in both control and SCO patient cells. These data suggest that the ability of SCO1 to interact with each of these proteins somehow facilitates the maturation of the CuA site of COX II

The core components of organelle biogenesis and membrane transport in the hydrogenosomes of Trichomonas vaginalis

Trichomonas vaginalis is a parasitic protist of the Excavata group. It contains an anaerobic form of mitochondria called hydrogenosomes, which produce hydrogen and ATP; the majority of mitochondrial pathways and the organellar genome were lost during the mitochondrion-to-hydrogenosome transition. Consequently, all hydrogenosomal proteins are encoded in the nucleus and imported into the organelles. However, little is known about the membrane machineries required for biogenesis of the organelle and metabolite exchange. Using a combination of mass spectrometry, immunofluorescence microscopy, in vitro import assays and reverse genetics, we characterized the membrane proteins of the hydrogenosome. We identified components of the outer membrane (TOM) and inner membrane (TIM) protein translocases include multiple paralogs of the core Tom40-type porins and Tim17/22/23 channel proteins, respectively, and uniquely modified small Tim chaperones. The inner membrane proteins TvTim17/22/23-1 and Pam18 were shown to possess conserved information for targeting to mitochondrial inner membranes, but too divergent in sequence to support the growth of yeast strains lacking Tim17, Tim22, Tim23 or Pam18. Full complementation was seen only when the J-domain of hydrogenosomal Pam18 was fused with N-terminal region and transmembrane segment of the yeast homolog. Candidates for metabolite exchange across the outer membrane were identified including multiple isoforms of the β-barrel proteins, Hmp35 and Hmp36; inner membrane MCF-type metabolite carriers were limited to five homologs of the ATP/ADP carrier, Hmp31. Lastly, hydrogenosomes possess a pathway for the assembly of C-tail-anchored proteins into their outer membrane with several new tail-anchored proteins being identified. These results show that hydrogenosomes and mitochondria share common core membrane components required for protein import and metabolite exchange; however, they also reveal remarkable differences that reflect the functional adaptation of hydrogenosomes to anaerobic conditions and the peculiar evolutionary history of the Excavata group

Compensatory renal hypertrophy, mitochondria and redox status

A reduction in functional renal mass occurs in humans during aging and severe kidney damage from diseases, injuries, infections and congenital conditions and after nephrectomy. Nephrectomy, or surgical removal of a kidney or a section of a kidney, is performed for treatment of unilateral secondary renal cancer, infections and for kidney transplantation. As a result, the remaining renal tissue undergoes compensatory growth due primarily to hypertrophy, in which both the size and functional capacity of the remaining kidney are increased. Renal compensatory hypertrophy is associated with a series of physiological, morphological and biochemical changes that also have toxicological implications. Previous studies have shown that compensatory renal cellular hypertrophy after uninephrectomy resulted in a hypermetabolic state, increased glutathione (GSH) content, but higher renal oxidative stress. These changes are also associated with increased susceptibility of renal proximal tubule cells to several drugs and environmental chemicals. Furthermore, our lab also showed that overexpression of mitochondrial GSH transporters, the dicarboxylate (DIC, Slc25a10) and 2-oxoglutarate (OGC, Slc25a11) carriers, in NRK-52E cells, which are derived from normal rat kidney proximal tubules, exhibited increased mitochondrial GSH uptake, contents of GSH and protection from chemically induced apoptosis from exposure to nephrotoxicants. Based on these previous observations, we hypothesized that compensatory renal hypertrophy after uninephrectomy alters renal function in vivo and mitochondrial status and modulation of mitochondrial redox status alters susceptibility to nephrotoxicants in the remnant kidney. In this study, we used a uninephrectomized (NPX) rat model to induce compensatory renal growth. Our results show alterations in renal physiological parameters consistent with modest renal injury, altered renal cellular energetics, upregulation of certain renal plasma membrane transporters, including some that have been observed to transport GSH, and evidence of increased oxidative stress in mitochondria from the remnant kidney of NPX rats. Our present results provide further evidence that compensatory renal hypertrophy is associated with mitochondrial hypertrophy and hyperpolarization and manipulation of mitochondrial GSH transporters in PT cells of hypertrophied kidney alters susceptibility to chemically induced injury. These studies provide additional insight into the molecular changes that occur in compensatory renal hypertrophy and should help in the development of novel therapeutic approaches for patients with reduced renal mass

Transportadores de fosfato e outros mecanismos adaptativos de plantas cultivadas em solos deficientes em fósforo.

O fósforo (P) é um elemento crucial para a existência das várias formas de vida na terra. Em todo o globo, o incremento da produção agrícola é um desafio constante para responder à demanda crescente da população mundial por alimento. Contudo, a maioria dos solos apresenta quantidades limitadas de P disponível, portanto, inadequados para a agricultura de alta performance. Tradicionalmente, o uso de adubo fosfatado encarece a produção e contamina o meio ambiente, quando aplicado em excesso, sendo incompatível com a implantação de modelos baseados em sustentabilidade. O documento aborda diferentes aspectos da adaptação de plantas e suas interações com organismos do solo nos desafios impostos pela quantidade limitada de P disponível e traz alguns resultados importantes das pesquisas realizadas nas últimas décadas pela equipe multidisciplinar da Embrapa Milho e Sorgo na busca de soluções, principalmente para a escassez de P disponível para as plantas cultivadas nos solos do Cerrado brasileiro