The Mitochondrial Inner Membrane Research Articles

Mitochondrial microproteins: critical regulators of protein import, energy production, stress response pathways, and programmed cell death.

Mitochondria rely upon the coordination of protein import, protein translation, and proper functioning of oxidative phosphorylation (OXPHOS) complexes I-V to sustain the activities of life for an organism. Each process is dependent upon the function of profoundly large protein complexes found in the mitochondria [translocase of the outer mitochondrial membrane (TOMM) complex, translocase of the inner mitochondrial membrane (TIMM) complex, OXPHOS complexes, mitoribosomes]. These massive protein complexes, in some instances more than one megadalton, are built up from numerous protein subunits of varying sizes, including many proteins that are ≤100-150 amino acids. However, these small proteins, termed microproteins, not only act as cogs in large molecular machines but also have important steps in inhibiting or promoting the intrinsic pathway of apoptosis, coordinate responses to cellular stress, and even act as hormones. This review focuses on microproteins that occupy the mitochondria and are critical for its function. Although the microprotein field is relatively new, researchers have long recognized the existence of these mitochondrial proteins as critical components of virtually all aspects of mitochondrial biology. Thus, recent studies estimating that hundreds of new microproteins of unknown function exist and are missing from current genome annotations suggests that the mitochondrial "microproteome" is a rich area for future biological investigation.

Blood and liver telomere length, mitochondrial DNA copy number, and hepatic gene expression of mitochondrial dynamics in mid-lactation cows supplemented with l-carnitine under systemic inflammation

The current study was conducted to examine the effect of l-carnitine (LC) supplementation on telomere length and mitochondrial DNA copy number (mtDNAcn) per cell in mid-lactation cows challenged by lipopolysaccharide (LPS) in blood and liver. The mRNA abundance of 31 genes related to inflammation, oxidative stress, and the corresponding stress response mechanisms, the mitochondrial quality control and the protein import system, as well as the phosphatidylinositol 3-kinase/protein kinase B pathway, were assessed using microfluidics integrated fluidic circuit chips (96.96 dynamic arrays). In addition to comparing the responses in cows with or without LC, our objectives were to characterize the oxidative and inflammatory status by assessing the circulating concentration of lactoferrin (Lf), haptoglobin (Hp), fibrinogen, derivates of reactive oxygen metabolites (dROM), and arylesterase activity (AEA), and to extend the measurement of Lf and Hp to milk. Pluriparous Holstein cows were assigned to either a control group (CON, n = 26) or an LC-supplemented group (CAR; 25 g LC/cow per day; d 42 ante partum to d 126 postpartum (PP), n = 27). On d 111 PP, each cow was injected intravenously with LPS (Escherichia coli O111:B4, 0.5 µg/kg). The mRNA abundance was examined in liver biopsies of d -11 and +1 relative to LPS administration. Plasma and milk samples were frequently collected before and after the challenge. After LPS administration, circulating plasma fibrinogen and serum dROM concentrations increased, whereas AEA decreased. Moreover, serum P4 initially increased by 3 h after LPS administration and declined thereafter irrespective of grouping. The Lf concentrations increased in both groups after LPS administration, with the CAR group showing greater concentrations in serum and milk than the CON group. After LPS administration, telomere length in blood increased, whereas mtDNAcn per cell decreased; however, both remained unaffected in liver. For mitochondrial protein import genes, the hepatic mRNA abundance of the translocase of the mitochondrial inner membrane (TIM)-17B was increased in CAR cows. Moreover, TIM23 increased in both groups after LPS administration. Regarding the mRNA abundance of genes related to stress response mechanisms, 7 out of 14 genes showed group × time interactions, indicating a (local) protective effect due to the dietary LC supplementation against oxidative stress in mid-lactating dairy cows. For mtDNAcn and telomere length, the effects of the LPS-induced inflammation were more pronounced than the dietary supplementation of LC. Dietary LC supplementation affected the response to LPS primarily by altering mitochondrial dynamics. Regarding mRNA abundance of genes related to the mitochondrial protein import system, the inner mitochondrial membrane translocase (TIM complex) seemed to be more sensitive to dietary LC than the outer mitochondrial membrane translocase (TOM complex).

Novel IM-associated protein Tim54 plays a role in the mitochondrial import of internal signal-containing proteins in Trypanosoma brucei.

The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus-encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS). Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex. TbTim54, a trypanosome-specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited the in vitro mitochondrial import and assembly of the internal signal-containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N-terminal signal-containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate-specificity of TbTim54 for internal-signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N-terminal domain of TbTim17. TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal-containing IM proteins in T.brucei. We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 in T.brucei.

The Cross Talk between TbTim50 and PIP39, Two Aspartate-Based Protein Phosphatases, Maintains Cellular Homeostasis in Trypanosoma brucei.

Trypanosoma brucei, the infectious agent of a deadly disease known as African trypanosomiasis, undergoes various stresses during its digenetic life cycle. We previously showed that downregulation of T. brucei mitochondrial inner membrane protein translocase 50 (TbTim50), an aspartate-based protein phosphatase and a component of the translocase of the mitochondrial inner membrane (TIM), increased the tolerance of procyclic cells to oxidative stress. Using comparative proteomics analysis and further validating the proteomics results by immunoblotting, here we discovered that TbTim50 downregulation caused an approximately 5-fold increase in the levels of PIP39, which is also an aspartate-based protein phosphatase and is primarily localized in glycosomes. A moderate upregulation of a number of glycosomal enzymes was also noticed due to TbTim50 knockdown. We found that the rate of mitochondrial ATP production by oxidative phosphorylation decreased and that substrate-level phosphorylation increased due to depletion of TbTim50. These results were correlated with relative increases in the levels of trypanosome alternative oxidase and hexokinase and a reduced-growth phenotype in low-glucose medium. The levels and activity of the mitochondrial superoxide dismutase and glutaredoxin levels were increased due to TbTim50 knockdown. Furthermore, we show that TbTim50 downregulation increased the cellular AMP/ATP ratio, and as a consequence, phosphorylation of AMP-activated protein kinase (AMPK) was increased. Knocking down both TbTim50 and TbPIP39 reduced PIP39 levels as well as AMPK phosphorylation and reduced T. brucei tolerance to oxidative stress. These results suggest that TbTim50 and PIP39, two protein phosphatases in mitochondria and glycosomes, respectively, cross talk via the AMPK pathway to maintain cellular homeostasis in the procyclic form of T. bruceiIMPORTANCETrypanosoma brucei, the infectious agent of African trypanosomiasis, must adapt to strikingly different host environments during its digenetic life cycle. Developmental regulation of mitochondrial activities is an essential part of these processes. We have shown previously that mitochondrial inner membrane protein translocase 50 in T. brucei (TbTim50) possesses a dually specific phosphatase activity and plays a role in the cellular stress response pathway. Using proteomics analysis, here we have elucidated a novel connection between TbTim50 and a protein phosphatase of the same family, PIP39, which is also a differentiation-related protein localized in glycosomes. We found that these two protein phosphatases cross talk via the AMPK pathway and modulate cellular metabolic activities under stress. Together, our results indicate the importance of a TbTim50 and PIP39 cascade for communication between mitochondria and other cellular parts in regulation of cell homeostasis in T. brucei.

The divergent N-terminal domain of Tim17 is critical for its assembly in the TIM complex in Trypanosoma brucei

Trypanosoma brucei Tim17(TbTim17), the single member of the Tim17/23/22 protein family, is an essential component of the translocase of the mitochondrial inner membrane (TIM). In spite of the conserved secondary structure, the primary sequence of TbTim17, particularly the N-terminal hydrophilic region, is significantly divergent. In order to understand the function of this region we expressed two N-terminal deletion mutants (Δ20 and Δ30) of TbTim17 in T. brucei. Both of these mutants of TbTim17 were targeted to mitochondria, however, they failed to complement the growth defect of TbTim17 RNAi cells. In addition, the import defect of other nuclear encoded proteins into TbTim17 knockdown mitochondria were not restored by expression of the N-terminal deletion mutants but complemented by knock-in of the full-length protein. Further analysis revealed that Δ20-TbTim17 and Δ30-TbTim17 mutants were not localized in the mitochondrial inner membrane. Analysis of the protein complexes in the wild type and mutant mitochondria by two-dimensional Blue-native/SDS-PAGE revealed that none of these mutants are assembled into the TbTim17 protein complex. However, FL-TbTim17 was integrated into the mitochondrial inner membrane and assembled into TbTim17 complex. Co-immunoprecipitation analysis showed that unlike the FL-TbTim17, mutant proteins are not associated with the endogenous TbTim17 as well as its interacting partner TbTim62, a novel trypanosome specific Tim. Together, these results show that the N-terminal domain of TbTim17 plays unique and essential roles for its sorting and assembly into the TbTim17 protein complex.

Tim62, a Novel Mitochondrial Protein in Trypanosoma brucei, Is Essential for Assembly and Stability of the TbTim17 Protein Complex

Trypanosoma brucei, the causative agent of human African trypanosomiasis, possesses non-canonical mitochondrial protein import machinery. Previously, we characterized the essential translocase of the mitochondrial inner membrane (TIM) consisting of Tim17 in T. brucei. TbTim17 is associated with TbTim62. Here we show that TbTim62, a novel protein, is localized in the mitochondrial inner membrane, and its import into mitochondria depends on TbTim17. Knockdown (KD) of TbTim62 decreased the steady-state levels of TbTim17 post-transcriptionally. Further analysis showed that import of TbTim17 into mitochondria was not inhibited, but its half-life was reduced >4-fold due to TbTim62 KD. Blue-native gel electrophoresis revealed that TbTim62 is present primarily in ∼150-kDa and also in ∼1100-kDa protein complexes, whereas TbTim17 is present in multiple complexes within the range of ∼300 to ∼1100 kDa. TbTim62 KD reduced the levels of both TbTim62 as well as TbTim17 protein complexes. Interestingly, TbTim17 was accumulated as lower molecular mass complexes in TbTim62 KD mitochondria. Furthermore, depletion of TbTim62 hampered the assembly of the ectopically expressed TbTim17-2X-myc into TbTim17 protein complex. Co-immunoprecipitation analysis revealed that association of TbTim17 with mHSP70 was markedly reduced in TbTim62 KD mitochondria. All together our results demonstrate that TbTim62, a unique mitochondrial protein in T. brucei, is required for the formation of a stable TbTim17 protein complex. TbTim62 KD destabilizes this complex, and unassembled TbTim17 degrades. Therefore, TbTim62 acts as a novel regulatory factor to maintain the levels of TIM in T. brucei mitochondria.

NMR investigations of the dual targeting peptide of Thr‐tRNA synthetase and its interaction with the mitochondrial Tom20 receptor in Arabidopsis thaliana

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins containing an N-terminal targeting peptide and are imported into mitochondria through the import machineries, the translocase of the outer mitochondrial membrane (TOM) and the translocase of the inner mitochondrial membrane (TIM). The N-terminal targeting peptide of precursor proteins destined for the mitochondrial matrix is recognized by the Tom20 receptor and plays an important role in the import process. Protein import is usually organelle specific, but several plant proteins are dually targeted into mitochondria and chloroplasts using an ambiguous dual targeting peptide. We present NMR studies of the dual targeting peptide of Thr-tRNA synthetase and its interaction with Tom20 in Arabidopsis thaliana. Our findings show that the targeting peptide is mostly unstructured in buffer, with a propensity to form α-helical structure in one region, S6-F27, and a very weak β-strand propensity for Q34-Q38. The α-helical structured region has an amphiphilic character and a φχχφφ motif, both of which have previously been shown to be important for mitochondrial import. Using NMR we have mapped out two regions in the peptide that are important for Tom20 recognition: one of them, F9-V28, overlaps with the amphiphilic region, and the other comprises residues L30-Q39. Our results show that the targeting peptide may interact with Tom20 in several ways. Furthermore, our results indicate a weak, dynamic interaction. The results provide for the first time molecular details on the interaction of the Tom20 receptor with a dual targeting peptide.

Bioenergetics of mitochondria of the liver with biliary atresia during prolonged starvation

L.),a representative of the class Cyclostomata, is a uniqueobject for studying the bioenergetics of liver mitochon-dria not only in the theoretical aspect but also in termsof clinical studies for the following reasons. Duringanadromous migrations in rivers (in our case, in theNeva River), lampreys switch off exogenous nutrition.This period, called synchrony, continues from Septem-ber to May. In May–June, lampreys as monocyclic ani-mals spawn and then die. Thus, cyclostomes provide arare opportunity to study the physiology and biochem-istry of the liver during multi-month starvation. In addi-tion, the liver of an adult lamprey lacks the gallbladderand bile ducts; as a result of biliary atresia (a fatal dis-ease of newborns), lamprey hepatocytes accumulatebile acids and bile pigments (bilirubin, biliverdin, andhemosiderin) [1]. It is known that cholestasis in humansis provoked by many drugs and is the result of manyliver pathologies [2]. In addition, during winter starva-tion, hepatocytes of lampreys (especially those ofmales) are filled with lipid drops [3]. The combinationof hepatic steatosis and cholestasis (steatocholestasis isa genetic metabolic liver disease widespread amongchildren [4]) makes lamprey liver an ideal naturalmodel for studying the bioenergetic of mitochondria inthese conditions, which are severe pathologies inhumans and a quite natural phase of the life cycle ofcyclostomes. During anadromous migration, lamprey liver almostdoes not contain glycogen, liver metabolism at this timeis solely anaerobic and is based on fatty acid oxidation[5]. Earlier, we showed that, after three- to four-monthstarvation in winter, the hepatocyte respiration of lam-preys reduces several times compared to the level char-acteristic of early migrants in autumn and in theprespawning period in spring [6]. The content of ade-nine nucleotides (in particular, ATP) in winter drasti-cally decreases, precisely corresponding to the respira-tory activity of hepatocytes. In approximately 25% ofexamined lampreys, the Atkinson energy charge (EC,[ATP] + 1/2 [ADP]/[ATP + ADP + AMP]) drops to crit-ically low values (0.2–0.3), which is characteristic ofcells in anabiosis, which was first described by us forhepatocytes of a vertebrate animal [7]. In the majorityof cells, EC was 0.5–0.6. At this potential value, cellscould survive but were incapable of growth and repro-duction. The goal of this work was to study the bioen-ergetic of isolated mitochondrial fractions of the lam-prey in winter during metabolic depression.Mitochondrial fractions were isolated in sucrose bythe conventional procedure [3]. In some cases, the iso-lation and incubation media were supplemented withbovine serum albumin (BSA). The mitochondrial respi-ration rates were measured with an LP-7 polarographequipped with a Clark electrode. The potential on theinner mitochondrial membrane was assessed bychanges in the fluorescence intensity (arb. units) of saf-ranine in a mitochondrial suspension with a ShimadzuRF-1501 spectrofluorometer (Shimadzu, Japan) at anexcitation wavelength of 485 nm and an emissionwavelength of 590 nm (Fig. 1). Mitochondria (0.5 mgprotein/ml) were added to a quartz cell filled an incuba-tion medium (3 ml) containing 250 mM sucrose, 3 mMTris-HCl (pH 7.3), 3 mM MgCl

Conserved substrate binding by chaperones in the bacterial periplasm and the mitochondrial intermembrane space

Mitochondria were derived from intracellular bacteria and the mitochondrial intermembrane space is topologically equivalent to the bacterial periplasm. Both compartments contain ATP-independent chaperones involved in the transport of hydrophobic membrane proteins. The mitochondrial TIM (translocase of the mitochondrial inner membrane) 10 complex and the periplasmic chaperone SurA were examined in terms of evolutionary relation, structural similarity, substrate binding specificity and their function in transporting polypeptides for insertion into membranes. The two chaperones are evolutionarily unrelated; structurally, they are also distinct both in their characteristics, as determined by SAXS (small-angle X-ray scattering), and in pairwise structural comparison using the distance matrix alignment (DALILite server). Despite their structural differences, SurA and the TIM10 complex share a common binding specificity in Pepscan assays of substrate proteins. Comprehensive analysis of the binding on a total of 1407 immobilized 13-mer peptides revealed that the TIM10 complex, like SurA, does not bind hydrophobic peptides generally, but that both chaperones display selectivity for peptides rich in aromatic residues and with net positive charge. This common binding specificity was not sufficient for SurA to completely replace TIM10 in yeast cells in vivo. In yeast cells lacking TIM10, when SurA is targeted to the intermembrane space of mitochondria, it binds translocating substrate proteins, but fails to completely transfer the substrate to the translocase in the mitochondrial inner membrane. We suggest that SurA was incapable of presenting substrates effectively to the primitive TOM (translocase of the mitochondrial outer membrane) and TIM complexes in early mitochondria, and was replaced by the more effective small Tim chaperone.

Characterization of thePreprotein and Amino Acid TransporterGene Family in Arabidopsis

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.

Probing the membrane topology of a subunit of the mitochondrial protein translocase, Tim44, with biotin maleimide

Tim44 is an essential component of the translocase of the inner mitochondrial membrane (TIM) complex that mediates transport of nuclear encoded mitochondrial precursors across the inner membrane. Here, we have investigated the topology of Tim44 by probing mitochondria with membrane impermeable 3-( N-maleimidopropionyl)biocytin (MPB) followed by the specific immunoprecipitation of modified proteins. Our data indicate that a single cysteine residue, Cys-369, located in the C-terminal domain of the yeast Tim44 is exposed to the mitochondrial intermembrane space.

The Mitochondrial Import Machinery for Preproteins

Most mitochondrial proteins are transported from the cytosol into the or-ganelle. Due to the division of mitochondria into an outer and inner membrane, an inter-membrane space and a matrix, an elaborated system for recognition and transport of preproteins has evolved. The translocase of the outer mitochondrial membrane (TOM) and the translocases of the inner mitochondrial membrane (TIM) mediate these processes. Receptor proteins on the cytosolic face of mitochondria recognize the cargo proteins and transfer them to the general import pore (GIP) of the outer membrane. Following the passage of preproteins through the outer membrane they are transported with the aid of the TIM23 complex into either the matrix, inner membrane, or intermembrane space. Some preprotein families utilize the TIM22 complex for their insertion into the inner membrane. The identification of protein components, which are involved in these transport processes, as well as significant insights into the molecular function of some of them, has been achieved in recent years. Moreover, we are now approaching a new era in which elaborated techniques have already allowed and will enable us to gather information about the TOM and TIM complexes on an ultrastructural level.