Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane

Translocation of proteins across the mitochondrial inner membrane is an essential process requiring an import motor having mitochondrial Hsp70 (mtHsp70) at its core. The J protein partner of mtHsp70, Pam18, is an integral part of this motor, serving to stimulate the ATPase activity of mtHsp70. Pam16, an essential protein having an inactive J domain that is unable to stimulate mtHsp70's ATPase activity, forms a heterodimer with Pam18, but its function is unknown. We set out to test the importance of three properties of Pam16: (i) a stable interaction between Pam16 and Pam18, (ii) the inability of Pam16's degenerate J domain to stimulate Ssc1's ATPase domain, and (iii) the innately lower stimulatory activity of the Pam16:Pam18 heterodimer, compared to Pam18 alone. Neither substantial reduction in the ability of Pam18 to stimulate Ssc1's ATPase activity, nor the presence of an active J domain in Pam16, had deleterious effects on cell growth, indicating the lack of importance of two of these biochemical properties. However, a stable interaction between Pam16's degenerate J domain and Pam18's J domain was found to be critical for function. Alterations that destabilized the Pam16:Pam18 heterodimer had deleterious effects on cell growth and mitochondrial protein import; intragenic suppressors that restored robust growth also restored heterodimer stability. Our results support the idea that Pam16's J-like domain strongly interacts with Pam18's J domain, leading to a productive interaction of Pam18 with mtHsp70 at the import channel.     

Substrate specificity of the TIM22 mitochondrial import pathway revealed with small molecule inhibitor of protein translocation

The TIM22 protein import pathway mediates the import of membrane proteins into the mitochondrial inner membrane and consists of two intermembrane space chaperone complexes, the Tim9-Tim10 and Tim8-Tim13 complexes. To facilitate mechanistic studies, we developed a chemical-genetic approach to identify small molecule agonists that caused lethality to a tim10-1 yeast mutant at the permissive temperature. One molecule, MitoBloCK-1, attenuated the import of the carrier proteins including the ADP/ATP and phosphate carriers, but not proteins that used the TIM23 or the Mia40/Erv1 translocation pathways. MitoBloCK-1 impeded binding of the Tim9-Tim10 complex to the substrate during an early stage of translocation, when the substrate was crossing the outer membrane. As a probe to determine the substrate specificity of the small Tim proteins, MitoBloCK-1 impaired the import of Tim22 and Tafazzin, but not Tim23, indicating that the Tim9-Tim10 complex mediates the import of a subset of inner membrane proteins. MitoBloCK-1 also inhibited growth of mammalian cells and import of the ADP/ATP carrier, but not TIM23 substrates, confirming that MitoBloCK-1 can be used to understand mammalian mitochondrial import and dysfunction linked to inherited human disease. Our approach of screening chemical libraries for compounds causing synthetic genetic lethality to identify inhibitors of mitochondrial protein translocation in yeast validates the generation of new probes to facilitate mechanistic studies in yeast and mammalian mitochondria.     

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.     

The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast.

We have cloned the gene encoding the protein Mas22p, which spans the outer membrane of yeast mitochondria. Cells that completely lack Mas22p are inviable. The plasmid-borne MAS22 gene suppresses several defects resulting from the deletion of one or more of the mitochondrial protein import receptors. Defects of Mas20p-deficient cells are explained by the reduced level of Mas22p in these mutants. Mas22p has one acidic domain in the cytosol and a second acidic domain in the mitochondrial intermembrane space. We suggest that these domains of Mas22p on either side of the outer membrane function as a relay system for transferring the basic targeting sequences of precursor proteins into the mitochondria.     

The dimensions of the protein import channels in the outer and inner mitochondrial membranes

Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins. We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 A and 26 A during translocation. The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel. These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.     

An intermediate step in translocation of lipopolysaccharide to the outer membrane of Salmonella typhimurium.

Evidence for transient localization of newly synthesized lipopolysaccharide at the periplasmic face of the inner membrane has been obtained by immunoelectron microscopic techniques. Salmonella typhimurium galE mutants in which O-antigen synthesis is dependent on addition of exogenous galactose were employed, and the distribution and fate of pulse-synthesized O antigen was examined by indirect ferritin labeling with anti-O-antigen IgG of spheroplasts prepared by treatment with lysozyme/EDTA. O-reactive lipopolysaccharide appeared rapidly at the exposed periplasmic face of the inner membrane after addition of galactose and was rapidly depleted upon termination of the pulse. Control experiments showed that secondary redistribution of lipopolysaccharide from outer membrane did not occur under the conditions employed for spheroplast formation and immunolabeling, and the pulse-chase kinetics were consistent with those expected for an intermediate in translocation of lipopolysaccharide to the outer membrane. In addition, undecaprenol-linked O antigen was detectable at the periplasmic face of the inner membrane within 30 sec after addition of galactose to a galE deep rough double mutant, and it accumulated stably in that location. The mutation in synthesis of the lipopolysaccharide core in the deep rough strain prevents transfer of O-antigen chains from undecaprenol phosphate to lipopolysaccharide. The result suggests that attachment of O antigen to lipopolysaccharide occurs on the extracytoplasmic side of the inner membrane and supports the conclusion that lipopolysaccharide is translocated to the outer membrane from the periplasmic, rather than the cytoplasmic, face of the inner membrane.     

J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18, encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.     

Translocation of a folded protein across the outer membrane in Escherichia coli.

A mutation in the Escherichia coli dsbA gene (coding for a periplasmic disulfide oxidoreductase) reduces the rate of disulfide bond formation in the enzyme pullulanase and also reduces the rate at which the enzyme is secreted to the cell surface, as measured by protease accessibility. The enzyme did not become protease accessible when disulfide bond formation was completely prevented in the mutant strain by carboxymethylation. These results indicate that a disulfide bond may be required for, and certainly does not impede, the translocation of pullulanase across the outer membrane. Since it is unlikely that a disulfide bond could be formed and then reduced again in the periplasm, these results would appear to strengthen the argument that pullulanase polypeptides fold into or close to their final conformation before they are transported across the outer membrane. It is suggested that this might be a feature common to all proteins that are secreted by other Gram-negative bacteria by a pullulanase-like pathway.     

Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane

Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain (“passenger domain”) and a C-terminal β barrel domain (“β domain”). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP–BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the β domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction.     

A discrete water exit pathway in the membrane protein cytochrome c oxidase

By using the non-redox-active Mg2+/Mn2+ site of cytochrome c oxidase as a probe, water access from the outside of the enzyme and water escape from the buried active site were studied. Water movement was time-resolved by monitoring the magnetic interaction of the oxygen isotope 17O with the Mn2+ by using a rapid freeze-quench-electron spin echo envelope modulation technique. Rapid (msec) access of water from the bulk phase to the Mn2+ was demonstrated by mixing cytochrome c oxidase with H217O. To determine whether a channel involving the Mn2+ was used for water exit from the active site, samples incubated in 17O2 were allowed to turn over approximately five times before freezing. The 17O, now in the form of H217O, was detected at the Mn2+. The significant broadening of the Mn2+ signal after the limited number of turnovers strongly suggests that the water exits the protein by means of one discrete pathway, not by random diffusion.     

Identification of a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane.

Import of proteins into mitochondria involves the cooperation of protein translocation systems in the outer and inner membranes. We have identified a 45-kDa protein at the protein import site of the yeast mitochondrial inner membrane. This 45-kDa protein could be crosslinked to a partly translocated precursor, which cannot be imported across the inner membrane when the matrix is depleted of ATP. In addition, an antibody against this protein strongly inhibited protein import into right-side-out inner-membrane vesicles. The 45-kDa protein accounts for only 0.1% of mitochondrial protein and appears peripherally attached to the outer face of the inner membrane. The properties of this protein suggest that it is a component of the protein import system of the mitochondrial inner membrane.     

Dynamic interaction between Isp45 and mitochondrial hsp70 in the protein import system of the yeast mitochondrial inner membrane.

The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp70 (mhsp70) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[gamma-thio]triphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp70 and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp70 and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.     

Translocation of proteins across the mitochondrial inner membrane, but not into the outer membrane, requires nucleoside triphosphates in the matrix.

Work in several laboratories has established that import of proteins across the mitochondrial inner membrane requires an electrochemical potential across that membrane and cleavage of nucleoside triphosphate. We now show that nucleoside triphosphate must be present inside the inner membrane. In contrast, the potential-independent insertion of porin into the outer membrane requires ATP only outside the inner membrane.     

Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6-f complex: position of the cytochrome b hemes in the membrane.

The amino acid sequences of cytochrome b of complex III from five different mitochondrial sources (human, bovine, mouse, yeast, and Aspergillus nidulans) and the chloroplast cytochrome b6 from spinach show a high degree of homology. Calculation of the distribution of hydrophobic residues with a "hydropathy" function that is conserved in this family of proteins implies that the membrane-folding pattern of the 42-kilodalton (kDa) mitochondrial cytochromes involves 8-9 membrane-spanning domains. The smaller 23-kDa chloroplast cytochrome appears to fold in five spanning domains that are similar to the first five of the mitochondria. Four highly conserved histidines are considered to be the likely ligands for the two hemes. The positions of the histidines along the spanning segments and in a cross section of the membrane-spanning alpha helices implies that two ligand pairs, His-82-His-197/198 and His-96-His-183, bridge the spanning peptides II and V, and the two hemes reside on opposite sides of the hydrophobic membrane core. In addition, the 17-kDa protein of the chloroplast b6-f complex appears to contain one or more of the functions of the COOH-terminal end of the mitochondrial cytochrome b polypeptide.     

Carboxyl-terminal sequences influence the import of mitochondrial protein precursors in vivo.

The large subunit of carbamoyl phosphate synthase A [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] from Neurospora crassa is encoded by a nuclear gene but is localized in the mitochondrial matrix. We have utilized N. crassa strains that produce both normal and carboxyl-terminal-truncated forms of carbamoyl phosphate synthase A to ask whether the carboxyl terminus affects import of the carbamoyl phosphate synthase A precursor. We found that carboxyl-terminal-truncated precursors were directed to mitochondria but that they were imported less efficiently than full-length proteins that were synthesized in the same cytoplasm. Our results suggest that effective import of proteins into mitochondria requires appropriate combinations of targeting sequences and three-dimensional structure.     

Early disturbance of calcium translocation across the plasma membrane in toxic liver injury

An increased influx and/or a decreased extrusion of calcium across the plasma membrane resulting in an increase in cytosolic-free calcium could play an important role in the initiation of irreversible cell injury. Therefore, the translocation of calcium across the plasma membrane was probed in the perfused rat liver using multiple indicator dilution methodology. The sucrose space corresponding to the extracellular space amounted to 0.35 +/- 0.13 ml per gm liver, and the water space corresponding to the extra- and intracellular spaces was 0.97 +/- 0.08 ml per gm. The calcium space was always slightly larger (0.42 +/- 0.10 ml per gm) than the sucrose space. The calcium space further increased during perfusion with the calcium ionophore A 23187, whereas the sucrose space remained unchanged. Two hours after administration to intact rats of acetaminophen (2 gm per kg) and carbon tetrachloride (2 ml per kg), respectively, the calcium space had increased markedly relative to the sucrose space and relative to the water space, indicating an increased accessibility of the cells to extracellular calcium. Similarly, reperfusion of livers after 90 min of ischemia was associated with an increase in calcium space relative to the sucrose and water spaces. These studies indicate that, in three models of acute liver injury, the net influx of calcium across the plasma membrane is increased early in the evolution of the injury before irreversible damage occurs.     

Lateral translational diffusion of cytochrome c oxidase in the mitochondrial energy-transducing membrane

The degree of freedom for lateral translational diffusion by cytochrome c oxidase and other integral proteins in the energy-transducing membrane of the mitochondrion was determined by combining the use of an immunoglobulin probe monospecific for the oxidase with thermotropic lipid phase transitions. Lateral mobility of the oxidase was monitored by observing the distribution of the immunoglobulin probe on the membrane surface by deep-etch electron microscopy and by observing the distribution of intramembrane particles (integral proteins) in the hydrophobic interior of the membrane by freeze-fracture electron microscopy. Incubation of the membrane with the immunoglobulin resulted in a time-dependent clustering of predominantly large intramembrane particles. Low temperature-induced lipid phase transitions resulted in the close packing of all intramembrane particles and cytochrome c oxidase by lateral exclusion from domains of gel-state bilayer lipid and was completely reversible. However, when cytochrome c oxidase was crosslinked through an immunoglobulin lattice prior to returning the membrane to above the lipid phase transition temperature, small intramembrane particles rerandomized while the large oxidase-related particles remained clustered. These observations reveal that cytochrome c oxidase can diffuse laterally in the energy-transducing membrane, either independently of all other integral proteins or in physical union with one or more other integral proteins. In addition, many other as yet unidentified smaller integral proteins can diffuse independently of the oxidase.     

Location of protein(s) involved in oligomycin-induced inhibition of mitochondrial adenosinetriphosphatase near the outer surface of the inner membrane

Mitoplasts, that is, mitochondria freed from their outer membranes, were prepared from pig heart. Sonication induced an inversion of these mitoplasts, giving inside-out vesicles. Added cytochrome c can be bound much better to mitoplasts than to sonicated vesicles; addition of trypsin increased adenosinetriphosphatase (ATPase) (ATP phosphohydrolase; EC 3.6.1.3) activity of sonicated vesicles without significantly affecting that of the mitoplasts. Since the site of fixation of cytochrome c was located on the outer side of the inner mitochondrial membrane and since the protein inhibitor of the mitochondrial ATPase is present on the inner face of the inner membrane and is very sensitive to trypsin, it can be concluded that mitoplasts are mainly oriented as normal mitochondria while sonicated vesicles are mainly inverted.Trypsin treatment can abolish the oligomycin sensitivity of ATPase activity of either mitoplasts or sonicated vesicles. However, trypsin induced the solubilization of the soluble F(1)-ATPase of sonicated vesicles while the ATPase activity remained with the mitoplasts after trypsin action. Therefore, trypsin destroyed the oligomycin effect by rupturing the liaison between F(1) and the membrane in sonicated vesicles. On the other hand, the effect of trypsin on mitoplasts must be attributed to the hydrolysis of a protein located near the outer surface of the inner membrane that is at least structurally involved in the oligomycin sensitivity of the ATPase complex.     

The heteroplasmic 15059G>A mutation in the mitochondrial cytochrome b gene and essential hypertension in type 2 diabetes

AimThe long-term stress of high blood pressure levels increases the risk of a variety of macro- and microvascular complications of type 2 diabetes (T2D). The etiology of essential hypertension (EH) has been explored in depth, but the pathophysiology is multifactorial, complex, and poorly understood. Recent findings showed a role of inherited mutations in mitochondrial DNA (mtDNA) in maternally inherited forms of hypertension. However, an impact of somatic mtDNA mutations in the development of EH is significantly less investigated. In this study, we examined whether the level of heteroplasmy for the 15059G>A mutation in the mitochondrial cytochrome b gene is associated with EH in T2D.Patients and methodsThe heteroplasmy level in mtDNA isolated from blood of 189 diabetic participants randomly selected from general population (124 of whom had EH) was quantified using a real-time PCR.ResultsThe 15059G>A heteroplasmy exceeding 39% was found to be significantly associated with a higher risk of EH (odds ratio 1.96; P (Fisher) 0.032).ConclusionThere is the first evidence reporting association between the mtDNA 15059G>A mutation heteroplasmy and EH in T2D.