Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F₁-ATPase α-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F₁-ATPase α-subunit.     

The zinc-binding protein Hot13 promotes oxidation of the mitochondrial import receptor Mia40

A disulphide relay system mediates the import of cysteine-containing proteins into the intermembrane space of mitochondria. This system consists of two essential proteins, Mia40 and Erv1, which bind to newly imported proteins by disulphide transfer. A third component, Hot13, was proposed to be important in the biogenesis of cysteine-rich proteins of the intermembrane space, but the molecular function of Hot13 remained unclear. Here, we show that Hot13, a conserved zinc-binding protein, interacts functionally and physically with the import receptor Mia40. It improves the Erv1-dependent oxidation of Mia40 both in vivo and in vitro. As a consequence, in mutants lacking Hot13, the import of substrates of Mia40 is impaired, particularly in the presence of zinc ions. In mitochondria as well as in vitro, Hot13 can be functionally replaced by zinc-binding chelators. We propose that Hot13 maintains Mia40 in a zinc-free state, thereby facilitating its efficient oxidation by Erv1.     

Erv1 mediates the Mia40-dependent protein import pathway and provides a functional link to the respiratory chain by shuttling electrons to cytochrome c

Unlike matrix-targeted or inner membrane proteins, those that are targeted to the mitochondrial intermembrane space (IMS) do not require ATP or the inner membrane electrochemical potential. Their import is mediated primarily by the essential IMS protein Mia40/Tim40. Here, we show that the mitochondrial flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidase Erv1 (essential for respiration and vegetative growth 1) plays a central role in the biogenesis of small, cysteine proteins of the IMS that are import substrates for Mia40. In a temperature-sensitive strain of Erv1, steady-state levels of small translocases of the inner membrane (Tims) are specifically affected when cells are grown at the non-permissive temperature. Furthermore, mitochondria isolated from the erv1-ts show a specific import and assembly defect for the small Tims but not in any other protein import pathway. Erv1 does not directly oxidise the small Tims, as thiol trapping assays show that the small Tims can still be oxidised in erv1-ts cells grown at the non-permissive temperature and in isolated mitochondria from this strain. Moreover, addition of pure Erv1 into erv1-ts mitochondria lacking the endogenous protein restores import and assembly of the small Tims only to an extent, arguing for a cascade of interactions with Erv1 rather than for a direct interaction of Erv1 with the small Tims. Cytochrome c (cyt c) is the in vivo oxidase for Erv1, as yeast cells mutated in cyt c cannot grow under anaerobic conditions. Therefore, Erv1 functionally links the Mia40-dependent import pathway to the Mia40-independent cyt c import pathway transferring electrons from the incoming precursors to cyt c as an acceptor. In this context, the protein import process is linked to the respiratory chain via the communication of Erv1 with cyt c.     

Presequence-dependent folding ensures MrpL32 processing by the m-AAA protease in mitochondria

m-AAA proteases exert dual functions in the mitochondrial inner membrane: they mediate the processing of specific regulatory proteins and ensure protein quality control degrading misfolded polypeptides to peptides. Loss of these activities leads to neuronal cell death in several neurodegenerative disorders. However, it is unclear how the m-AAA protease chooses between specific processing and complete degradation. A central and conserved function of the m-AAA protease is the processing of the ribosomal subunit MrpL32, which regulates ribosome biogenesis and the formation of respiratory complexes. Here, we demonstrate that the formation of a tightly folded domain harbouring a conserved CxxC-X(9)-CxxC sequence motif halts degradation initiated from the N-terminus and triggers the release of mature MrpL32. Oxidative stress impairs folding of MrpL32, resulting in its degradation by the m-AAA protease and decreased mitochondrial translation. Surprisingly, MrpL32 folding depends on its mitochondrial targeting sequence. Presequence-assisted folding of MrpL32 requires the complete import of the MrpL32 precursor before maturation occurs and therefore explains the need for post-translocational processing by the m-AAA protease rather than co-translocational cleavage by the general mitochondrial processing peptidase.     

The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.     

Mia40 Protein Serves as an Electron Sink in the Mia40-Erv1 Import Pathway

A redox-regulated import pathway consisting of Mia40 and Erv1 mediates the import of cysteine-rich proteins into the mitochondrial intermembrane space. Mia40 is the oxidoreductase that inserts two disulfide bonds into the substrate simultaneously. However, Mia40 has one redox-active cysteine pair, resulting in ambiguity about how Mia40 accepts numerous electrons during substrate oxidation. In this study, we have addressed the oxidation of Tim13 in vitro and in organello. Reductants such as glutathione and ascorbate inhibited both the oxidation of the substrate Tim13 in vitro and the import of Tim13 and Cmc1 into isolated mitochondria. In addition, a ternary complex consisting of Erv1, Mia40, and substrate, linked by disulfide bonds, was not detected in vitro. Instead, Mia40 accepted six electrons from substrates, and this fully reduced Mia40 was sensitive to protease, indicative of conformational changes in the structure. Mia40 in mitochondria from the erv1–101 mutant was also trapped in a completely reduced state, demonstrating that Mia40 can accept up to six electrons as substrates are imported. Therefore, these studies support that Mia40 functions as an electron sink to facilitate the insertion of two disulfide bonds into substrates.     

线粒体蛋白质运输机制

：线粒体的大多数蛋白质是由核基因编码、细胞质合成,而最终运输到线粒体。在此过程中,需要线粒体外膜和内膜的蛋白质运输机器(至少三种主要的移位酶复合物)来保证前体蛋白质的正确运输。     

Active remodelling of the TIM23 complex during translocation of preproteins into mitochondria

The TIM23 (translocase of the mitochondrial inner membrane) complex mediates translocation of preproteins across and their insertion into the mitochondrial inner membrane. How the translocase mediates sorting of preproteins into the two different subcompartments is poorly understood. In particular, it is not clear whether association of two operationally defined parts of the translocase, the membrane-integrated part and the import motor, depends on the activity state of the translocase. We established conditions to in vivo trap the TIM23 complex in different translocation modes. Membrane-integrated part of the complex and import motor were always found in one complex irrespective of whether an arrested preprotein was present or not. Instead, we detected different conformations of the complex in response to the presence and, importantly, the type of preprotein being translocated. Two non-essential subunits of the complex, Tim21 and Pam17, modulate its activity in an antagonistic manner. Our data demonstrate that the TIM23 complex acts as a single structural and functional entity that is actively remodelled to sort preproteins into different mitochondrial subcompartments.     

An atlas of chaperone–protein interactions in Saccharomyces cerevisiae: implications to protein folding pathways in the cell

Molecular chaperones are known to be involved in many cellular functions, however, a detailed and comprehensive overview of the interactions between chaperones and their cofactors and substrates is still absent. Systematic analysis of physical TAP‐tag based protein–protein interactions of all known 63 chaperones in Saccharomyces cerevisiae has been carried out. These chaperones include seven small heat‐shock proteins, three members of the AAA+ family, eight members of the CCT/TRiC complex, six members of the prefoldin/GimC complex, 22 Hsp40s, 1 Hsp60, 14 Hsp70s, and 2 Hsp90s. Our analysis provides a clear distinction between chaperones that are functionally promiscuous and chaperones that are functionally specific. We found that a given protein can interact with up to 25 different chaperones during its lifetime in the cell. The number of interacting chaperones was found to increase with the average number of hydrophobic stretches of length between one and five in a given protein. Importantly, cellular hot spots of chaperone interactions are elucidated. Our data suggest the presence of endogenous multicomponent chaperone modules in the cell.     

Protein folding in the secretory pathway of animal cells

The exit of newly-synthesized proteins from the lumen of the endoplasmic reticulum (ER) is the rate-determining step in protein secretion. Only correctly-folded and fully-assembled proteins exit the ER and progress along the secretory pathway. Folding and assembly in the ER are mediated by a variety of factors including folding catalysts and molecular chaperones. The properties of these factors, and the nature of their interactions with folding substrates, are beginning to be clarified. Little work has been done to characterize these processes and these factors in cell lines employed for large-scale cell culture. Manipulation of these process may permit improvement in yield or productivity of recombinant proteins by cultured animal cells.     

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19-Cys43 disulfide bond that links the beta-sheet core with the alpha-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2A resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by approximately 1.5 kcal mol(-1) (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity.     

Sequential steps and checkpoints in the early exocytic compartment during secretory IgM biogenesis

The biogenesis of secretory IgM occurs stepwise under stringent quality control, formation of μ₂L₂ preceding polymerization. How is efficiency of IgM secretion coupled to fidelity? We show here that ERp44, a soluble protein involved in thiol-mediated retention, interacts with ERGIC-53. Binding to this hexameric lectin contributes to ERp44 localization in the ER-golgi intermediate compartment. ERp44 and ERGIC-53 increase during B-lymphocyte differentiation, concomitantly with the onset of IgM polymerization. Both preferentially bind μ₂L₂ and higher order intermediates. Their overexpression or silencing in non-lymphoid cells promotes or decreases secretion of IgM polymers, respectively. In IgM-secreting B-lymphoma cells, μ chains interact first with BiP and later with ERp44 and ERGIC-53. Our findings suggest that ERGIC-53 provides a platform that receives μ₂L₂ subunits from the BiP-dependent checkpoint, assisting polymerization. In this process, ERp44 couples thiol-dependent assembly and quality control.     

The therapeutic potential of chemical chaperones in protein folding diseases

Several neurodegenerative diseases are caused by defects in protein folding, including Alzheimer, Parkinson, Huntington, and prion diseases. Once a disease-specific protein misfolds, it can then form toxic aggregates which accumulate in the brain, leading to neuronal dysfunction, cell death, and clinical symptoms. Although significant advances have been made toward understanding the mechanisms of protein aggregation, there are no curative treatments for any of these diseases. Since protein misfolding and the accumulation of aggregates are the most upstream events in the pathological cascade, rescuing or stabilizing the native conformations of proteins is an obvious therapeutic strategy. In recent years, small molecules known as chaperones have been shown to be effective in reducing levels of misfolded proteins, thus minimizing the accumulation of aggregates and their downstream pathological consequences. Chaperones are classified as molecular, pharmacological, or chemical. In this mini-review we summarize the modes of action of different chemical chaperones and discuss evidence for their efficacy in the treatment of protein folding diseases in vitro and in vivo.     

Age‐dependent cardiomyopathy in mitochondrial mutator mice is attenuated by overexpression of catalase targeted to mitochondria

Mitochondrial defects have been found in aging and several age‐related diseases. Mice with a homozygous mutation in the exonuclease encoding domain of mitochondrial DNA polymerase gamma (Polg^m/m) are prone to age‐dependent accumulation of mitochondrial DNA mutations and have shown a broad spectrum of aging‐like phenotypes. However, the mechanism of cardiac phenotypes in relation to the role of mitochondrial DNA mutations and oxidative stress in this mouse model has not been fully addressed. We demonstrate age‐dependent cardiomyopathy in Polg^m/m mice, which by 13–14 months of age displays marked cardiac hypertrophy and dilatation, impairment of systolic and diastolic function, and increased cardiac fibrosis. This age‐dependent cardiomyopathy is associated with increases in mitochondrial DNA (mtDNA) deletions and protein oxidative damage, increased expression of apoptotic and senescence markers, as well as a decline in signaling for mitochondrial biogenesis. The relationship of these changes to mitochondrial reactive oxygen species (ROS) was tested by crossing Polg^m/m mice with mice that overexpress mitochondrial targeted catalase (mCAT). All of the above phenotypes were partially rescued in Polg^m/m/mCAT mice. These data indicate that accumulation of mitochondrial DNA damage with age can lead to cardiomyopathy and that this phenotype is partly mediated by mitochondrial oxidative stress.     

The endoplasmic reticulum of plant cells and its role in protein maturation and biogenesis of oil bodies

The endoplasmic reticulum (ER) is the port of entry of proteins into the endomembrane system, and it is also involved in lipid biosynthesis and storage. This organelle contains a number of soluble and membrane-associated enzymes and molecular chaperones, which assist the folding and maturation of proteins and the deposition of lipid storage compounds. The regulation of translocation of proteins into the ER and their subsequent maturation within the organelle have been studied in detail in mammalian and yeast cells, and more recently also in plants. These studies showed that in general the functions of the ER in protein synthesis and maturation have been highly conserved between the different organisms. Yet, the ER of plants possesses some additional functions not found in mammalian and yeast cells. This compartment is involved in cell to cell communication via the plasmodesmata, and, in specialized cells, it serves as a storage site for proteins. The plant ER is also equipped with enzymes and structural proteins which are involved in the process of oil body biogenesis and lipid storage. In this review we discuss the components of the plant ER and their function in protein maturation and biogenesis of oil bodies. Due to the large number of cited papers, we were not able to cite all individual references and in many cases we refer the readers to reviews and references therein. We apologize to the authors whose references are not cited.     

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein,Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.     

Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.     

On the accuracy of metadynamics and its variations in a protein folding process

Metadynamics and its variations are powerful tools for exploring the free energy landscape of physical, chemical and biophysical systems. However, previous tests of their accuracy were based on either low-dimensional systems or complicated systems without exact answers. Therefore their accuracy, particularly when used for high-dimensional biophysical systems, has not been rigorously tested. In this work we performed a series of simulations with metadynamics and its variations for a typical biophysical process – the folding of protein chymotrypsin inhibitor 2 (CI2) based on a coarse-grained structure-based model. The results revealed the power as well as limitations of the algorithms and provided some guidelines for using metadynamics and its variations in high-dimensional biological systems.