Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): Evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry

Homologues of Drosophilia transient receptor potential (TRP) have been proposed to be unitary subunits of plasma membrane ion channels that are activated as a consequence of active or passive depletion of Ca2+ stores. In agreement with this hypothesis, cells expressing TRPs display novel Ca2+-permeable cation channels that can be activated by the inositol 1,4,5-trisphosphate receptor (IP3R) protein. Expression of TRPs alters cells in many ways, including up-regulation of IP3Rs not coded for by TRP genes, and proof that TRP forms channels of these and other cells is still missing. Here, we document physical interaction of TRP and IP3R by coimmunoprecipitation and glutathione S-transferase-pulldown experiments and identify two regions of IP3R, F2q and F2g, that interact with one region of TRP, C7. These interacting regions were expressed in cells with an unmodified complement of TRPs and IP3Rs to study their effect on agonist- as well as store depletion-induced Ca2+ entry and to test for a role of their respective binding partners in Ca2+ entry. C7 and an F2q-containing fragment of IP3R decreased both forms of Ca2+ entry. In contrast, F2g enhanced the two forms of Ca2+ entry. We conclude that store depletion-activated Ca2+ entry occurs through channels that have TRPs as one of their normal structural components, and that these channels are directly activated by IP3Rs. IP3Rs, therefore, have the dual role of releasing Ca2+ from stores and activating Ca2+ influx in response to either increasing IP3 or decreasing luminal Ca2+.

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.

Effect of age on in vivo rates of mitochondrial protein synthesis in human skeletal muscle

A progressive decline in muscle performance in the rapidly expanding aging population is causing a dramatic increase in disability and health care costs. A decrease in muscle endurance capacity due to mitochondrial decay likely contributes to this decline in muscle performance. We developed a novel stable isotope technique to measure in vivo rates of mitochondrial protein synthesis in human skeletal muscle using needle biopsy samples and applied this technique to elucidate a potential mechanism for the age-related decline in the mitochondrial content and function of skeletal muscle. The fractional rate of muscle mitochondrial protein synthesis in young humans (24 ± 1 year) was 0.081 ± 0.004%·h−1, and this rate declined to 0.047 ± 0.005%·h−1 by middle age (54 ± 1 year; P < 0.01). No further decline in the rate of mitochondrial protein synthesis (0.051 ± 0.004%·h−1) occurred with advancing age (73 ± 2 years). The mitochondrial synthesis rate was about 95% higher than that of mixed protein in the young, whereas it was approximately 35% higher in the middle-aged and elderly subjects. In addition, decreasing activities of mitochondrial enzymes were observed in muscle homogenates (cytochrome c oxidase and citrate synthase) and in isolated mitochondria (citrate synthase) with increasing age, indicating declines in muscle oxidative capacity and mitochondrial function, respectively. The decrease in the rates of mitochondrial protein synthesis is likely to be responsible for this decline in muscle oxidative capacity and mitochondrial function. These changes in muscle mitochondrial protein metabolism may contribute to the age-related decline in aerobic capacity and muscle performance.

Chemical chaperones mediate increased secretion of mutant α1-antitrypsin (α1-AT) Z: A potential pharmacological strategy for prevention of liver injury and emphysema in α1-AT deficiency

In α1-AT deficiency, a misfolded but functionally active mutant α1-ATZ (α1-ATZ) molecule is retained in the endoplasmic reticulum of liver cells rather than secreted into the blood and body fluids. Emphysema is thought to be caused by the lack of circulating α1-AT to inhibit neutrophil elastase in the lung. Liver injury is thought to be caused by the hepatotoxic effects of the retained α1-ATZ. In this study, we show that several “chemical chaperones,” which have been shown to reverse the cellular mislocalization or misfolding of other mutant plasma membrane, nuclear, and cytoplasmic proteins, mediate increased secretion of α1-ATZ. In particular, 4-phenylbutyric acid (PBA) mediated a marked increase in secretion of functionally active α1-ATZ in a model cell culture system. Moreover, oral administration of PBA was well tolerated by PiZ mice (transgenic for the human α1-ATZ gene) and consistently mediated an increase in blood levels of human α1-AT reaching 20–50% of the levels present in PiM mice and normal humans. Because clinical studies have suggested that only partial correction is needed for prevention of both liver and lung injury in α1-AT deficiency and PBA has been used safely in humans, it constitutes an excellent candidate for chemoprophylaxis of target organ injury in α1-AT deficiency.

Human deafness dystonia syndrome is a mitochondrial disease

The human deafness dystonia syndrome results from the mutation of a protein (DDP) of unknown function. We show now that DDP is a mitochondrial protein and similar to five small proteins (Tim8p, Tim9p, Tim10p, Tim12p, and Tim13p) of the yeast mitochondrial intermembrane space. Tim9p, Tim10p, and Tim12p mediate the import of metabolite transporters from the cytoplasm into the mitochondrial inner membrane and interact structurally and functionally with Tim8p and Tim13p. DDP is most similar to Tim8p. Tim8p exists as a soluble 70-kDa complex with Tim13p and Tim9p, and deletion of Tim8p is synthetically lethal with a conditional mutation in Tim10p. The deafness dystonia syndrome thus is a novel type of mitochondrial disease that probably is caused by a defective mitochondrial protein-import system.

Preferential Zn2+ influx through Ca2+-permeable AMPA/kainate channels triggers prolonged mitochondrial superoxide production

Synaptically released Zn2+ can enter and cause injury to postsynaptic neurons. Microfluorimetric studies using the Zn2+-sensitive probe, Newport green, examined levels of [Zn2+]i attained in cultured cortical neurons on exposure to N-methyl-d-asparte, kainate, or high K+ (to activate voltage-sensitive Ca2+ channels) in the presence of 300 μM Zn2+. Indicating particularly high permeability through Ca2+-permeable α-amino3-hydroxy-5-methyl-4-isoxazolepropionic-acid/kainate (Ca-A/K) channels, micromolar [Zn2+]i rises were observed only after kainate exposures and only in neurons expressing these channels [Ca-A/K(+) neurons]. Further studies using the oxidation-sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species (ROS) generation that paralleled the [Zn2+]i rises, with rapid oxidation observed only in the case of Zn2+ entry through Ca-A/K channels. Indicating a mitochondrial source of this ROS generation, hydroethidine oxidation was inhibited by the mitochondrial electron transport blocker, rotenone. Additional evidence for a direct interaction between Zn2+ and mitochondria was provided by the observation that the Zn2+ entry through Ca-A/K channels triggered rapid mitochondrial depolarization, as assessed by using the potential-sensitive dye tetramethylrhodamine ethylester. Whereas Ca2+ influx through Ca-A/K channels also triggers ROS production, the [Zn2+]i rises and subsequent ROS production are of more prolonged duration.

Effect of the protein import machinery at the mitochondrial surface on precursor stability

Many biological processes require proteins to undergo conformational changes at the surface of membranes. For example, some precursor proteins unfold at the surface of mitochondria and chloroplasts before translocation into the organelles, and toxins such as colicin A unfold to the molten globule state at bacterial surfaces before inserting into the cell membrane. It is commonly thought that the membrane surfaces and the associated protein machinery destabilize the substrate proteins and that this effect is required for membrane insertion or translocation. One of the best characterized translocation processes is protein import into mitochondria. By measuring the contributions of individual interactions within a model protein to its stability at the mitochondrial surface and in free solution, we show here that the mitochondrial surface neither induces the molten globule state in this protein nor preferentially destabilizes any type of interaction (e.g., hydrogen bonds, nonpolar, etc.) within the protein. Because it is not possible to measure absolute protein stability at the surface of mitochondria, we determined the stability of a tightly associated protein–protein complex at the mitochondrial import site as a model of the stability of a protein. We found the binding constants of the protein–protein complex at the mitochondrial surface and in free solution to be identical. Our results demonstrate that the mitochondrial surface does not destabilize importing precursor proteins in its vicinity.

Essential role of Glu-C66 for menaquinol oxidation indicates transmembrane electrochemical potential generation by Wolinella succinogenes fumarate reductase

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone, in a reaction opposite to that catalyzed by the related enzyme succinate:quinone reductase (succinate dehydrogenase). In the previously determined structure of QFR from Wolinella succinogenes, the site of fumarate reduction in the flavoprotein subunit A of the enzyme was identified, but the site of menaquinol oxidation was not. In the crystal structure, the acidic residue Glu-66 of the membrane spanning, diheme-containing subunit C lines a cavity that could be occupied by the substrate menaquinol. Here we describe that, after replacement of Glu-C66 with Gln by site-directed mutagenesis, the resulting mutant is unable to grow on fumarate and the purified enzyme lacks quinol oxidation activity. X-ray crystal structure analysis of the Glu-C66 → Gln variant enzyme at 3.1-Å resolution rules out any major structural changes compared with the wild-type enzyme. The oxidation-reduction potentials of the heme groups are not significantly affected. We conclude that Glu-C66 is an essential constituent of the menaquinol oxidation site. Because Glu-C66 is oriented toward a cavity leading to the periplasm, the release of two protons on menaquinol oxidation is expected to occur to the periplasm, whereas the uptake of two protons on fumarate reduction occurs from the cytoplasm. Thus our results indicate that the reaction catalyzed by W. succinogenes QFR generates a transmembrane electrochemical potential.

Protein kinase A-catalyzed phosphorylation of heat shock protein 60 chaperone regulates its attachment to histone 2B in the T lymphocyte plasma membrane

Accumulating evidence suggests that the mitochondrial molecular chaperone heat shock protein 60 (hsp60) also can localize in extramitochondrial sites. However, direct evidence that hsp60 functions as a chaperone outside of mitochondria is presently lacking. A 60-kDa protein that is present in the plasma membrane of a human leukemic CD4+ CEM-SS T cell line and is phosphorylated by protein kinase A (PKA) was identified as hsp60. An 18-kDa plasma membrane-associated protein coimmunoprecipitated with hsp60 and was identified as histone 2B (H2B). Hsp60 physically associated with H2B when both molecules were in their dephospho forms. By contrast, PKA-catalyzed phosphorylation of both hsp60 and H2B caused dissociation of H2B from hsp60 and loss of H2B from the plasma membrane of intact T cells. These results suggest that (i) hsp60 and H2B can localize in the T cell plasma membrane; (ii) hsp60 functions as a molecular chaperone for H2B; and (iii) PKA-catalyzed phosphorylation of both hsp60 and H2B appears to regulate the attachment of H2B to hsp60. We propose a model in which phosphorylation/dephosphorylation regulates chaperoning of H2B by hsp60 in the plasma membrane.

Multiple independent origins of mitochondrial gene order in birds

Mitochondrial genomes of all vertebrate animals analyzed to date have the same 37 genes, whose arrangement in the circular DNA molecule varies only in the relative position of a few genes. This relative conservation suggests that mitochondrial gene order characters have potential utility as phylogenetic markers for higher-level vertebrate taxa. We report discovery of a mitochondrial gene order that has had multiple independent originations within birds, based on sampling of 137 species representing 13 traditionally recognized orders. This provides evidence of parallel evolution in mitochondrial gene order for animals. Our results indicate operation of physical constraints on mitochondrial gene order changes and support models for gene order change based on replication error. Bird mitochondria have a displaced OL (origin of light-strand replication site) as do various other Reptilia taxa prone to gene order changes. Our findings point to the need for broad taxonomic sampling in using mitochondrial gene order for phylogenetic analyses. We found, however, that the alternative mitochondrial gene orders distinguish the two primary groups of songbirds (order Passeriformes), oscines and suboscines, in agreement with other molecular as well as morphological data sets. Thus, although mitochondrial gene order characters appear susceptible to some parallel evolution because of mechanistic constraints, they do hold promise for phylogenetic studies.

A role for intracellular pH in membrane IgM-mediated cell death of human B lymphomas

We show that anti-IgM-induced cell death in a human B lymphoma cell line, B104, is associated with early intracellular acidification and cell shrinkage. In contrast, another human B cell lymphoma line, Daudi, less susceptible to B cell antigen receptor-mediated cell death, responded to anti-IgM with an early increase in intracellular pH (pHi). The anti-IgM-induced changes of pHi were associated with different levels of activation of the Na+/H+ exchanger isoform 1 (NHE1) as judged by its phosphorylation status. Prevention of anti-IgM-induced cell death in B104 cells by the calcineurin phosphatase inhibitor, cyclosporin A, abrogated both intracellular acidification and cell shrinkage and was associated with an increase in the phosphorylation level of NHE1 within the first 60 min of stimulation. This indicates a key role for calcineurin in regulating pHi and cell viability. The potential role of pHi in cell viability was confirmed in Daudi cells treated with an Na+/H+ exchanger inhibitor 5-(N,N-hexamethylene)amiloride. These observations indicate that the outcome of the anti-IgM treatment depends on NHE1-controlled pHi. We suggest that inactivation of the NHE1 in anti-IgM-stimulated cells results in intracellular acidification and subsequently triggers or amplifies cell death.

Identification, Separation, and Characterization of Acyl-Coenzyme A Dehydrogenases Involved in Mitochondrial β-Oxidation in Higher Plants1

The existence in higher plants of an additional β-oxidation system in mitochondria, besides the well-characterized peroxisomal system, is often considered controversial. Unequivocal demonstration of β-oxidation activity in mitochondria should rely on identification of the enzymes specific to mitochondrial β-oxidation. Acyl-coenzyme A dehydrogenase (ACAD) (EC 1.3.99.2,3) activity was detected in purified mitochondria from maize (Zea mays L.) root tips and from embryonic axes of early-germinating sunflower (Helianthus annuus L.) seeds, using as the enzyme assay the reduction of 2,6-dichlorophenolindophenol, with phenazine methosulfate as the intermediate electron carrier. Subcellular fractionation showed that this ACAD activity was associated with mitochondrial fractions. Comparison of ACAD activity in mitochondria and acyl-coenzyme A oxidase activity in peroxisomes showed differences of substrate specificities. Embryonic axes of sunflower seeds were used as starting material for the purification of ACADs. Two distinct ACADs, with medium-chain and long-chain substrate specificities, respectively, were separated by their chromatographic behavior, which was similar to that of mammalian ACADs. The characterization of these ACADs is discussed in relation to the identification of expressed sequenced tags corresponding to ACADs in cDNA sequence analysis projects and with the potential roles of mitochondrial β-oxidation in higher plants.

Changes of Mitochondrial Properties in Maize Seedlings Associated with Selection for Germination at Low Temperature. Fatty Acid Composition, Cytochrome c Oxidase, and Adenine Nucleotide Translocase Activities1

Mitochondria are affected by low temperature during seedling establishment in maize (Zea mays L.). We evaluated the associated changes in the mitochondrial properties of populations selected for high (C4-H) and low (C4-L) germination levels at 9.5°C. When seedlings of the two populations were grown at 14°C (near the lower growth limit), the mitochondrial inner membranes of C4-H showed a higher percentage of 18-carbon unsaturated fatty acids, a higher fluidity, and a higher activity of cytochrome c oxidase. We found a positive relationship between these properties and the activity of a mitochondrial peroxidase, allowing C4-H to reduce lipid peroxidation relative to C4-L. The specific activity of reconstituted ATP/ADP translocase was positively associated with this peroxidase activity, suggesting that translocase activity is also affected by chilling. The level of oxidative stress and defense mechanisms are differently expressed in tolerant and susceptible populations when seedlings are grown at a temperature near the lower growth limit. Thus, the interaction between membrane lipids and cytochrome c oxidase seems to play a key role in maize chilling tolerance. Furthermore, the divergent-recurrent selection procedure apparently affects the allelic frequencies of genes controlling such an interaction.