Novel inter- and intrasubunit contacts between transport-relevant residues of the homodimeric mitochondrial phosphate transport protein

Ser158 is located near the middle of the matrix loop connecting transmembrane helices C and D of the mitochondrial phosphate transport protein (PTP). The mutant Ser158Thr PTP is transport-inactive. His32 is located near the middle of transmembrane helix A and Thr79 is located 5 residues away from transmembrane helix B and its N-terminal (matrix end). Single site mutant PTPs that have either residue replaced with Ala are transport-inactive. Based on the high resolution structure of a subunit of the bovine ADP/ATP translocase, on sequence similarities between members of the mitochondrial transport protein family, and on the PTP subunit/subunit contact site between transmembrane A helices, it is now suggested that the Ser158 site is at the PTP subunit/subunit contact site. This contact site is essential for keeping the transport cycles catalyzed by the two PTP subunits 180 degrees out of phase. The data also suggest that His32 and Thr79 of the same subunit interact and couple the phosphate and the proton transport paths.     

High-level bacterial expression of mitochondrial transport proteins

The inability to obtain abundant quantities of purified, functional membrane proteins has represented a significant impediment to the goal of obtaining high-resolution structural information. In this review, procedures are described which have been developed in this laboratory and enable the high-level bacterial expression and subsequent purification of functional mitochondrial citrate transport proteins from yeast and rat liver. The data that we have obtained using these procedures and related results from other laboratories are discussed. Additionally, the general applicability of this approach to most mitochondrial transport proteins as well as to other types of membrane proteins is considered. Finally, relevant considerations when contemplating the use of this methodology and the likely value of this approach in future areas of research are explored.     

Mitochondrial cation transport: A progress report

This report summarizes recent work in our laboratory aimed at understanding protein-mediated mitochondrial cation transport. We are studying three distinct cation cycles that contain porters catalyzing influx and efflux of cations between cytosol and mitochondrial matrix. Each of these cation cycles plays a major physiological role in the overall energy economy. The K⁺ cycle maintains the integrity of the vesicular structure and includes the K⁺/H⁺ antiporter, the K_ATP channel, and K⁺ leak driven by the high membrane potential. The Ca²⁺ cycle relays the signals calling for modulation of ATP production and includes the Ca²⁺ channel, the Na⁺/Ca²⁺ antiporter, and the Na⁺/H⁺ antiporter. The H⁺ cycle of brown adipose tissue mitochondria provides heat to hibernating and newborn mammals and consists of the uncoupling protein, which catalyzes regulated H⁺ influx.     

The human mitochondrial transport protein family: Identification and protein regions significant for transport function and substrate specificity

Protein sequence similarities and predicted structures identified 75 mitochondrial transport proteins (37 subfamilies) from among the 28,994 human RefSeq (NCBI) protein sequences. All, except two, have an E-value of less than 4e−05 with respect to the structure of the single subunit bovine ADP/ATP carrier/carboxyatractyloside complex (bAAC/CAT) (mGenThreader program). The two 30-kDa exceptions have E-values of 0.003 and 0.005. 21 have been functionally identified and belong to 14 subfamilies. A subset of subfamilies with sequence similarities for each of 12 different protein regions was identified. Many of the 12 protein regions for each tested protein yielded different size subsets. The sum of subfamilies in the 12 subsets was lowest for the phosphate transport protein (PTP) and highest for aralar 1. Transmembrane sequences are most unique. Sequence similarities are highest near the membrane center and matrix. They are highest for the region of transmembrane helices H1, H2 and connecting matrix loop 12 and smallest for transmembrane helices H3, H4 and loop 34. These sequence similarities and the predicted high similarities to the bAAC/CAT structure point to common structural/functional elements that could include subunit/subunit contact sites as they have been identified for PTP and AAC. The four residues protein segment (SerLysGlnIle) of loop 12 is the only segment projecting into the center of the funnel-like structure of the bAAC/CAT. It is present in its entirety only in the AACs and with some replacements in the large Ca2+-modulated aspartate/glutamate transporters. Other transporters have deletions and replacements in this region of loop 12. This protein segment with its central location and variation in size and composition likely contributes to the substrate specificity of the transporters.     

Simple models for the analysis of binding protein-dependent transport systems.

Mathematical modeling was used to evaluate experimental data for bacterial binding protein-dependent transport systems. Two simple models were considered in which ligand-free periplasmic binding protein interacts with the membrane-bound components of transport. In one, this interaction was viewed as a competition with the ligand-bound binding protein, whereas in the other, it was considered to be a consequence of the complexes formed during the transport process itself. Two sets of kinetic parameters were derived for each model that fit the available experimental results for the maltose system. By contrast, a model that omitted the interaction of ligand-free binding protein did not fit the experimental data. Some applications of the successful models for the interpretation of existing mutant data are illustrated, as well as the possibilities of using mutant data to test the original models and sets of kinetic parameters. Practical suggestions are given for further experimental design.     

Functional properties of purified and reconstituted mitochondrial metabolite carriers

Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties. A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This unphysiological transport mode is characterized by a combination of channel-type and carrier-type properties.     

Aquaporin-8-facilitated mitochondrial ammonia transport

Aquaporin-8 (AQP8) is a membrane channel permeable to water and ammonia. As AQP8 is expressed in the inner mitochondrial membrane of several mammalian tissues, we studied the effect of the AQP8 expression on the mitochondrial transport of ammonia. Recombinant rat AQP8 was expressed in the yeast Saccharomyces cerevisiae. The presence of AQP8 in the inner membrane of yeast mitochondria was demonstrated by subcellular fractionation and immunoblotting analysis. The ammonia transport was determined in isolated mitochondria by stopped flow light scattering using formamide as ammonia analog. We found that the presence of AQP8 increased by threefold mitochondrial formamide transport. AQP8-facilitated mitochondrial formamide transport in rat native tissue was confirmed in liver (a mitochondrial AQP8-expressing tissue) vs. brain (a mitochondrial AQP8 non-expressing tissue). Comparative studies indicated that the AQP8-mediated mitochondrial movement of formamide was markedly higher than that of water. Together, our data suggest that ammonia diffusional transport is a major function for mitochondrial AQP8.     

Progress in the definition of a reference human mitochondrial proteome

Owing to the complexity of higher eukaryotic cells, a complete proteome is likely to be very difficult to achieve. However, advantage can be taken of the cell compartmentalization to build organelle proteomes, which can moreover be viewed as specialized tools to study specifically the biology and "physiology" of the target organelle. Within this frame, we report here the construction of the human mitochondrial proteome, using placenta as the source tissue. Protein identification was carried out mainly by peptide mass fingerprinting. The optimization steps in two-dimensional electrophoresis needed for proteome research are discussed. However, the relative paucity of data concerning mitochondrial proteins is still the major limiting factor in building the corresponding proteome, which should be a useful tool for researchers working on human mitochondria and their deficiencies.     

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction

The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000muM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.     

L-arabinose transport and the L-arabinose binding protein of escherichia coli

The active accumulation of L-arabinose by arabinose induced cultures of Escherichia coli is mediated by 2 independent transport mechanisms. One, specified by the gene locus araE, is membrane bound and possesses a relatively “low affinity.” The other, specified in part by the genetic locus araF, contains as a functional component the L-arabinose binding protein and functions with a “high affinity” for the substrate. The L-arabinose binding protein has been purified, partially characterized, crystallized, and sequenced.     

Characterization of a periplasmic protein related to sn-glycerol-3-phosphate transport in escherichia coli

The cold osmotic shock procedure releases a protein (GLPT) from the cell envelope of Escherichia coli that is related to the transport of sn-glycerol-3-phosphate in this organism. The evidence for this correlation is as follows: (1) GLPT is under the regulatory control of the glpR gene. (2) Some glpT mutants that were isolated as phosphonomycin resistant clones do not synthesize GLPT. Revertants of these mutants (growth on sn-glycerol 3-phosphate) again synthesize GLPT. (3) Some amber mutations in glpT reduce the amount of GLPT while suppressed strains produce normal amounts. (4) Transfer of a plasmid carrying the glpT genes into a strain lacking GLPT and sn-glycerol-3-phosphate transport restores both functions in the recipient. Transport and GLPT synthesis in the plasmid carrying strain are increased 2- to 3-fold over a fully induced wild-type strain, but appear to be constitutive. GLPT is a soluble protein of molecular weight 160,000 composed of 4 identical subunits. The 160,000 molecular weight complex is stable in 1% sodium dodecylsulfate at room temperature. Upon boiling in 1% sodium dodecylsulfate GLPT dissociates into its subunits. Likewise, 8 M urea at room temperature dissociates GLPT into its subunits. Dialysis of dissociated GLPT against phosphate or Tris-HCl buffer, pH 7.0, allows renaturation to the tetrameric form. The protein is acidic in nature (isoelectric point 4.4). In contrast to the typical transport-related periplasmic-binding proteins, no conditions could be found where pure GLPT exhibited binding activity toward its supposed substrate, sn-glycerol-3-phosphate. In vivo new appearance of transport activity for sn-glycerol-3-phosphate transport occurs only shortly before cell division. However, GLPT synthesis does not fluctuate during the cell cycle. The available evidence indicates a cell-division-dependent processing of GLPT in the cell envelope as a reason for the alteration in transport activity. Transport in whole cells is sensitive to the cold osmotic shock procedure, demonstrating the participation of an essential periplasmic component. However, isolated membrane vesicles that are devoid of periplasmic components, including GLPT, are fully active in sn-glycerol-3-phosphate transport. Therefore, we conclude that GLPT is essential in overcoming a diffusion barrier for sn-glycerol-3-phosphate established by the outer membrane. Attempts to isolate mutants that are transport negative in whole cells due to a defect in GLPT but are active in isolated membrane vesicles have failed so far. All GLPT mutants tested, whether or not they synthesize GLPT, are not active in isolated membrane vesicles. Iodination of whole cells with [¹²⁵I] followed by osmotic shock reveals that several shock-releasable proteins including GLPT become radioactively labeled. This indicates that some portions of GLPT are accessible to the external medium.     

Citrate uniport by the mitochondrial tricarboxylate carrier: a basis for a new hypothesis for the transport mechanism

The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate–citrate exchange, while HgCl₂ and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH₂ residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.     

Analysis of protein transport in the Brassica oleracea vasculature reveals protein-specific destinations

We investigated the vascular transport properties of exogenously applied proteins to Brassica oleracea plants and compared their delivery to various aerial parts of the plant with carboxy fluorescein (CF) dye. We identified unique properties for each protein. Alexafluor-tagged bovine serum albumin (Alexa-BSA) and Alexafluor-tagged Histone H1 (Alexa-Histone) moved slower than CF dye throughout the plant. Interestingly, Alexa-Histone was retained in the phloem and phloem parenchyma while Alexa-BSA moved into the apoplast. One possibility is that Alexa-Histone sufficiently resembles plant endogenous proteins and is retained in the vascular stream, while Alexa-BSA is exported from the cell as a foreign protein. Both proteins diffuse from the leaf veins into the leaf lamina. Alexa-BSA accumulated in the leaf epidermis while Alexa-Histone accumulated mainly in the mesophyll layers. Fluorescein-tagged hepatitis C virus core protein (fluorescein-HCV) was also delivered to B. oleracea plants and is larger than Alexa-BSA. This protein moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the vascular transfer of exogenous proteins in B. oleracea plants. Specific protein properties appear to determine their destination and transport properties within the phloem.     

Comparative analysis of protein transport in the N. benthamiana vasculature reveals different destinations.

We investigated the vascular transport of exogenously applied proteins and compared their delivery to various aerial parts of the plant with carboxy fluorescein dye. Alexafluor tagged bovine serum albumin (Alexa-BSA) moves at a low level to upper parts of the plant and unloads to the apoplast. Alexafluor tagged Histone H1 (Alexa-Histone) moves rapidly throughout the plant and is retained in the phloem and phloem parenchyma. Both Alexa-Histone and -BSA were exported from leaf veins class II and III but they unloaded completely into the leaf lamina with barely any residual fluorescence left inside the leaf veins. Fluorescein tagged hepatitis C virus core protein (fluorescein-HCV) moves more rapidly than BSA through the plant and was restricted to the leaf veins. Fluorescein-HCV failed to unload to the leaf lamina. These combined data suggest that there is not a single default pathway for the transfer of exogenous proteins through the plant. Specific protein properties appear to determine their destination and transport properties within the phloem.     

Mitochondrial protein synthesis: Figuring the fundamentals,complexities and complications,of mammalian mitochondrial translation

Mitochondrial protein synthesis is essential for all mammals, being responsible for providing key components of the oxidative phosphorylation complexes. Although only thirteen different polypeptides are made, the molecular details of this deceptively simple process remain incomplete. Central to this process is a non-canonical ribosome, the mitoribosome, which has evolved to address its unique mandate. In this review, we integrate the current understanding of the molecular aspects of mitochondrial translation with recent advances in structural biology. We identify numerous key questions that we will need to answer if we are to increase our knowledge of the molecular mechanisms underlying mitochondrial protein synthesis.     

A generic model of electron transport in mitochondria

In this paper, a simplified, generic model of mitochondrial metabolism is explored. In particular the following question is addressed: To what extent are phenomena observed in experiments and simulations of mitochondrial metabolism generic, in the sense that they must occur in all models with this basic structure? Of particular interest are the electron transport chain and oxidative phosphorylation, and how flux through the system and the redox states of intermediates respond to physiologically important stimuli. These stimuli include changes in substrate supply (NADH/FADH(2)), in oxygenation, and in membrane proton gradient/ATP demand. Analytical techniques are used to show that certain experimentally observed effects must occur in the generic model. These include the responses of both flux and redox states to changed substrate and oxygen concentrations. At the same time other effects, such as the responses of redox states to changes in proton gradient, are dependent on the details of the model, and are not common to every model with the same basic structure. The phenomenon of saturation in response to large inputs is also discussed.     

HIV-1 viral genes and mitochondrial apoptosis

The mitochondrion is an organelle that regulates various cellular functions including the production of energy and programmed cell death. Aberrant mitochondrial function is often concomitant with various cytopathies and medical disorders. The mitochondrial membrane plays a key role in the induction of cellular apoptosis, and its destabilization, as triggered by both intracellular and extracellular stimuli, results in the release of proapoptotic factors into the cytosol. Not surprisingly, proteins from the human immunodeficiency virus type 1 (HIV) have been implicated in exploiting this organelle to promote the targeted depletion of key immune cells, which assists in viral evasion of the immune system and contributes to the characteristic global immunodeficiency observed during progression of disease. Here we review the mechanisms by which HIV affects the mitochondrion, and suggest that various viral-associated genes may directly regulate apoptotic cell death.     

Anion transport in rat brain mitochondria: Fumarate uptake via the dicarboxylate carrier

Penetration of fumarate into rat brain mitochondria has been investigated, as required in brain ammoniogenesis. Mitochondria swell in ammonium fumarate and this swelling is increased by both Pi and malate. According to a carrier mediated process, fumarate translocation, which occurs in exchange with intramitochondrial malate or Pi shows saturation characteristics. By photometrically investigating the kinetics of fumarate/malate, fumarate/ Pi and malate/Pi exchanges, different Km values were obtained (10, 22 and 250 M, respectively), whereas no significant difference was found forV _max values (40 nmol NAD(P)⁺ reduced/min×mg protein). This suggests that fumarate and malate share a single carrier to enter mitochondria, namely the dicarboxylate carrier. Both comparison made of theV _max values and inhibiton studies exclude a fumarate translocation via either the tricarboxylate carrier, whose occurrence in brain is here demonstrated, or oxodicarboxylate carrier. Kinetic investigation of the dicarboxylate translocator shows the existence of thiol group/s and metal ion/s at or near the substrate binding sites. The experimental findings are discussed in the light of fumarate uptake in vivo in brain ammoniogenesis.Abbreviations AD.SUCC adenylsuccinate - ASP aspartate - BTA 1,2,3,-benzenetricarboxylate - CITR citrate - D-NAD deamino-NAD - PUM fumarate - GABA -aminobutyrate - GAP glyceraldehyde-3-phosphate - GAP-DH glyceraldheyde-3-phosphate dehydrogenase - GHBA -hydroxybutyrate - HEPES 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - OAA oxaloacetate - OG oxoglutarate - PEP phosphoenolpyruvate - 3-PG glycerate-3-phosphate - 3-PGP glycerate-1,3-diphosphate - PYR pyruvate - RBM rat brain mitochondria - RHM rat heart mitochondria - RKM rat kidney mitochondria - RLM rat liver mitochondria - SSA succinic semialdehyde     

Neofunctionalization within the Omp85 protein superfamily during chloroplast evolution

The Toc75 and OEP80 proteins reside in the chloroplast outer envelope membrane. Both are members of the Omp85 superfamily of β-barrel proteins, and both are essential in Arabidopsis plants with important roles throughout development. Toc75 forms the translocation channel of the TOC complex, which is responsible for importing nucleus-encoded proteins into chloroplasts, while the function of OEP80 remains uncertain. Deficiency of Toc75 in plants that have artificially reduced OEP80 levels suggests that the latter may be involved in the biogenesis of β-barrel proteins, in similar fashion to Omp85-related proteins in other systems. To elucidate the evolutionary relationship between the two proteins, we conducted a phylogenetic analysis using 48 sequences from diverse species. This indicated that Toc75 and OEP80 belong to sister groups in the Omp85 superfamily, and originate from a gene duplication in an ancient eukaryotic organism > 1.2 billion years ago. Our analysis also supports the notion that the Toc75 family has undergone a phase of neofunctionalization to accommodate the organelle’s newly acquired need to import proteins.