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© 2012 John Wiley & Sons A/S doi:10.1111/tra.12030
Disulfide Bond Formation: Sulfhydryl Oxidase ALR Controls Mitochondrial Biogenesis of Human MIA40 Malgorzata E. Sztolsztener1 , Anita Brewinska1 , Bernard Guiard2 and Agnieszka Chacinska1,∗ 1 International
Institute of Molecular and Cell Biology, Laboratory of Mitochondrial Biogenesis Warsaw, 02-109 Poland 2 Centre de Gen ´ etique ´ ´ Moleculaire, CNRS, Gif-sur-Yvette, 91190 France *Corresponding author: Agnieszka Chacinska, [email protected]
The conserved MIA pathway is responsible for the import and oxidative folding of proteins destined for the intermembrane space of mitochondria. In contrast to a wealth of information obtained from studies with yeast, the function of the MIA pathway in higher eukaryotes has remained enigmatic. Here, we took advantage of the molecular understanding of the MIA pathway in yeast and designed a model of the human MIA pathway. The yeast model for MIA consists of two critical components, the disulfide bond carrier Mia40 and sulfhydryl oxidase Erv1/ALR. Human MIA40 and ALR substituted for their yeast counterparts in the essential function for the oxidative biogenesis of mitochondrial intermembrane space proteins. In addition, the sulfhydryl oxidases ALR/Erv1 were found to be involved in the mitochondrial localization of human MIA40. Furthermore, the defective accumulation of human MIA40 in mitochondria underlies a recently identified disease that is caused by amino acid exchange in ALR. Thus, human ALR is an important factor that controls not only the ability of MIA40 to bind and oxidize protein clients but also the localization of human MIA40 in mitochondria. Key words: ALR/GFER, disease, MIA pathway, mitochondria, oxidative folding, protein biogenesis Received 20 June 2012, revised and accepted for publication 19 November 2012, uncorrected manuscript published online 27 November 2012, published online 16 December 2012
Mitochondria contain approximately 1000 (yeast) to 1500 (human) different proteins that are involved in numerous fundamental cellular processes (1–3). Thus, the proper biogenesis of mitochondrial proteins is critical for the normal performance and survival of cells (4–6). The proteins localized in the intermembrane space (IMS) of mitochondria constitute a functionally important part of the mitochondrial proteome. Subsequent to their synthesis on cytosolic ribosomes, a few proteins destined for the IMS utilize a classical import pathway that depends on the inner mitochondrial membrane potential and TIM23, the translocase of the inner mitochondrial membrane. The
import of this class of proteins is driven by a cleavable amino-terminal bipartite signal that consists of a presequence and transmembrane anchor (5,7,8). However, the large group of IMS proteins contain conserved cysteine residues. These cysteine residues are arranged in twin CX3 C or CX9 C motifs in the families of small Tim proteins or Cox17, respectively, and are involved in the formation of disulfide bonds (9–14). The cysteine-rich precursor proteins are targeted to the IMS in a reduced, importcompetent state via the mitochondrial intermembrane space assembly (MIA) pathway (15–19). The essential MIA machinery components are the disulfide bond carrier Mia40 (mitochondrial IMS and assembly pathway 40 kDa) (20–22) and the sulfhydryl oxidase Erv1 (essential for respiration and growth viability 1) (23–26). Mia40 serves as a receptor that recognizes and forms transient intermediate complexes with the incoming protein precursors via intermolecular disulfide bonds (27–31). As a result of thiol-disulfide exchange reactions catalyzed by MIA, precursor proteins are subsequently released from the intermediate complexes in the oxidized state with two disulfide bonds that stabilize their structure (25,32–37). Mia40 contains an essential CPC motif (Figure 1A) that constitutes a redox-active disulfide bond involved in the binding and oxidative folding of precursor proteins (25,34,38,39). The precursor’s oxidation leaves the CPC motif of Mia40 in the reduced state. For subsequent rounds of protein import and oxidation, reduced Mia40 is activated by the sulfhydryl oxidase Erv1. Erv1 oxidizes the CPC motif, thereby maintaining Mia40 in an oxidized, import-competent state (25,38,40–42). Mia40 and Erv1 are conserved within the eukaryotic kingdom. Human MIA40 (hMIA40), although much smaller, harbors a domain with six conserved cysteine residues that are arranged in the redox-active CPC motif and twin CX9 C motif (Figure 1A; 34,43). Human augmenter of liver regeneration (hALR/GFER) is a mammalian homolog of Erv1. Both proteins belong to the flavin adenine dinucleotide (FAD)-dependent sulfhydryl oxidase family and are capable of forming disulfide bonds de novo (44–47). Similar to its yeast counterpart, hALR contains the flexible N-terminal shuttle domain with an invariant CX2 C motif directly involved in hMIA40 oxidation (Figure 1B). The C-terminal domain of hALR shares approximately 40% sequence identity with Erv1 and contains a noncovalently bound FAD cofactor and four conserved cysteine residues. Two of the residues are arranged in CX2 C to form a redox centre in close proximity to FAD, and two of the residues form CX16 C motifs with the structural role (40–42,47–50). The similarities between lower and higher eukaryotic proteins suggest that the MIA pathway operates under universal principles. www.traffic.dk 309
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Figure 1: hMIA40 and b2 -hALR localize in the IMS of yeast mitochondria. A and B) Schematic representation of hMIA40/Mia40 and hALR/Erv1 is shown. C in black boxes, conserved cysteine residues; C in grey boxes, nonconserved cysteine residues; TM, transmembrane domain. C) Experimental work flow to generate the S. cerevisiae strains expressing human proteins via plasmid shuffling. 5-FOA, 5-fluoroorotic acid; gDNA, genomic DNA. D) Growth complementation of the ERV1 deletion mutant by hALR or b2 -hALR . Strains were subjected to consecutive 10-fold dilutions, spotted on the minimal media plates in the presence or absence of 5-FOA and grown for 4 days at 28◦ C. E) Analysis of the hMIA40 and b2 -hALR distribution in yeast mitochondria upon hypoosmotic swelling followed by separation into mitochondrial pellet (P) or soluble fraction (S). WT, wild-type strain.
Very little is known about the functions of the MIA pathway in higher eukaryotes at the cellular level. hMIA40 was shown to be involved in the biogenesis of IMS proteins, such as typical CX3 C or CX9 C substrates, and also a copper chaperone, CCS1, that is required for the mitochondrial localization of superoxide dismutase 1 (43,51). Despite the presence of conserved cysteine residues, hMIA40 differs significantly from its yeast counterpart. Mia40 from yeast contains a large N-terminal extension with a presequence and transmembrane signal that anchors mature Mia40 in the inner mitochondrial membrane (Figure 1A; 22,31). Therefore, Mia40 is imported into yeast mitochondria in an inner membrane potential-dependent manner via the TIM23 pathway. Mia40 from higher eukaryotes does not contain a bipartite mitochondrial signal. Thus, it is directed into mitochondria via the disulfide relay (i.e. MIA) pathway and is present in the IMS in a soluble form (43,52). Both hMIA40 and hALR can functionally replace single components (i.e. Mia40 and Erv1) in yeast growth tests (52,53). Thus, in addition to hMIA40, the function of hALR is also likely in the disulfide relay and biogenesis of IMS proteins. This hypothesis was supported by the identification of a rare autosomal recessive myopathy that results from an Arg194His substitution in hALR (54). The cells of affected individuals display various defects, including abnormal mitochondrial morphology, 310
decreased activity of respiratory complexes I, II and IV, and also reduced levels of cysteine-rich TIMM13 and COX17 proteins, suggesting impaired function of the MIA pathway in the biogenesis of IMS proteins (54). In this study, we sought to understand the role of human components of the MIA pathway, hMIA40 and hALR, in the cell. The function of the essential MIA pathway has been extensively studied and is best understood in yeast (20–33,38,39,41,52,53). Thus, we chose yeast to investigate the universal and organism-specific features of the human MIA pathway in the cellular environment. Furthermore, having established a yeast model of the human MIA pathway, we investigated the molecular basis of a disease associated with ALR dysfunction. We found that the sulfhydryl oxidase ALR/Erv1 plays a novel role in the MIA pathway by regulating the localization of hMIA40.
Results Human MIA40 and ALR substitute for the function of yeast Mia40 and Erv1 that is essential to life To determine the function of hMIA40 and hALR in the biogenesis of mitochondria, we expressed hMIA40 and hALR (GeneBank accession no. BC033775 and Traffic 2013; 14: 309–320
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NM_005262, respectively) in Saccharomyces cerevisiae strains that lacked the essential components of the MIA machinery, Mia40 and Erv1. We previously demonstrated that a lack of growth caused by chromosomal deletion of the MIA40 gene can be rescued by hMIA40 expression, but the resulting strain that bore hMIA40 was not further analyzed (52). To substitute Erv1 for its human ortholog, we replaced MIA40 for hMIA40 to the genomic locus, deleted ERV1 and subjected the resulting strain to a standard plasmid shuffling procedure with plasmids encoding Erv1/hALR (Figure 1C). The b2 -hALR fusion was generated by the attachment of the cytochrome b2 presequence to the N-terminus of hALR. The cells with hALR did not survive, whereas cells with b2 -hALR grew as well as the cells with Erv1 as a positive control (Figure 1D). Human ALR is likely not able to substitute for Erv1 because of inefficient targeting to yeast mitochondria. However, the presence of b2 -hALR sustained the viability of yeast that lacked Erv1. These results are consistent with previous findings (53). For further analyses, based on the procedure described above, we obtained two yeast strains that produce human proteins hMIA40/Erv1 and hMIA40/b2 -hALR instead of their essential yeast counterparts, Mia40 and Erv1. We began by assessing the mitochondrial localization of human proteins. Isolated mitochondria were subjected to hypoosmotic swelling that resulted in the rupture of the outer mitochondrial membrane and solution accessibility of the IMS proteins. The analysis of the supernatants revealed that hMIA40 is released from mitochondria upon swelling (Figure 1E, lanes 5–12) indicating that hMIA40 is present in the IMS in a soluble form, unlike its yeast counterpart attached to the inner membrane (Figure 1E, lanes 1–4). The bipartite targeting signal of cytochrome b2 consists of a proteolytically processed presequence and membrane anchor and directs fusion protein constructs into the IMS of mitochondria. Initially, the matrix processing peptidase cleaves a presequence, leading to the generation of the intermediate protein form. Inner membrane peptidase activity then results in the cleavage of a hydrophobic anchor and release of the mature form of the protein to the IMS (21,52). The processing of the b2 -hALR fusion construct was efficient and resulted in the generation of the mature form of hALR (m-b2 -hALR) predominantly and only a small pool of the membrane-attached intermediate form of protein (i-b2 -hALR) (Figure 1E, lanes 9 and 11; longer exposure). In contrast to the membrane-anchored intermediate form, the mature form of hALR was partially released upon the hypoosmotic swelling of isolated mitochondria, similarly to hMIA40 and other soluble IMS proteins (Figure 1E). A partial release of Erv1 was reported previously (33), suggesting a peripheral association of protein with the inner mitochondrial membrane. Mitochondrial proteins present in the membranes and matrix were not affected by the hypoosmotic treatment (Figure 1E). These results demonstrate that hMIA40 and b2 -hALR are localized in the IMS in yeast mitochondria where they are able to
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complement the lack of the essential functions of their yeast counterparts.
Function of human MIA40 in protein import to the IMS is evolutionarily conserved Both hMIA40/Erv1 and hMIA40/b2 -hALR strains were analyzed for growth. Remarkably, the yeast in which two essential components, Mia40 and Erv1, were substituted with hMIA40 and b2 -hALR grew equally well as the wild-type strain under all conditions tested (Figure 2A). However, the hMIA40/Erv1 strain displayed an inhibition of growth at a higher temperature (36◦ C) on both fermentable and respiratory media and only slightly impaired growth at a lower temperature (Figure 2A). To gain more insights into the role of human proteins of the MIA pathway in mitochondrial biogenesis, we isolated mitochondria and performed the following experiments. First, we analyzed the mitochondria from yeast grown at a lower temperature in the fermentable medium. The levels of hMIA40 were slightly reduced in the Erv1 mitochondria compared with the b2 -hALR mitochondria (Figure 2B, lanes 3–6). However, the reduction in the amount of hMIA40 did not substantially influence the steady-state levels of the IMS proteins that utilize the MIA pathway (Figure 2B, lanes 7–12) or the proteins from other mitochondrial compartments (Figure 2B, lanes 13–18). We also studied the import of precursors into isolated mitochondria using the representatives of two classical substrate families of the MIA pathway, the CX3 C (small Tim proteins) and CX9 C (Cox19) families. The import experiments showed that hMIA40/Erv1 and hMIA40/b2 -hALR mitochondria imported Cox19, Tim13 and Tim9 into a proteaseprotected location with similar efficiency compared to wild-type mitochondria (Figure 2C). Mia40 forms disulfidebonded intermediates with the incoming precursors that are detected by nonreducing sodium dodecyl sulfate (SDS) electrophoresis (Figure 2D, lanes 9–12; 27,29,32,33). In mitochondria with hMIA40/Erv1 and hMIA40/b2 -hALR, the hMIA40 intermediate complexes were efficiently formed (Figure 2D, lanes 1–8). We wondered whether the same observations hold true for yeast grown in the respiratory medium (i.e. with a higher demand for mitochondrial function and biogenesis) at a lower temperature. Under these conditions, mitochondria with hMIA40/Erv1 and hMIA40/b2 -hALR contained equal levels of hMIA40 and other mitochondrial proteins (Figure S1A). This was accompanied by equal efficiency of the IMS proteins mitochondrial import (Figure S1B), hMIA40 intermediate complex formation and Tim13 oxidation (Figure S1C). The only exception was Erv1, which appeared to be increased in hMIA40/Erv1 compared with wild-type (Figure S1A, lanes 1–4). This putative compensatory increase in Erv1 may explain the lack of any detectable changes in the levels of hMIA40 in the hMIA40/Erv1 cells grown under respiratory conditions (Figure S1) compared with small defects observed for yeast grown under fermentative conditions (Figure 2). This 311
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Figure 2: hMIA40 and hALR function in the biogenesis of mitochondrial IMS proteins. A) Yeast strains were subjected to consecutive 10-fold dilutions, spotted onto YPG and YPS plates and grown for 5 days at the indicated temperatures. B) Mitochondria isolated from the strains grown in YPS medium at 19◦ C were analyzed by reducing SDS-PAGE followed by Western blotting. C and D) Isolated mitochondria were subjected to the import assays by incubation with radiolabeled Cox19, Tim13 and Tim9 precursors. As a control, import reactions were blocked by 50 mM IAA. Samples were separated by reducing (C) or nonreducing (D) SDS-PAGE followed by autoradiography. WT, wild-type strain.
analysis led us to conclude that if cells are not challenged by growth conditions (i.e. a higher growth temperature), then hMIA40 is fully capable of importing and oxidizing IMS proteins and cooperates well with both human (ALR) and yeast (Erv1) sulfhydryl oxidase in a disulfide relay. Altogether, we reconstituted the human MIA pathway in a heterologous system (i.e. yeast cells). We determined the conditions under which the presence of hALR is necessary for the proper functioning of this pathway.
Human ALR is required for mitochondrial biogenesis of human MIA40 A tendency to lose hMIA40 (Figure 2B) could underlie the growth defect of hMIA40/Erv1 at a higher temperature (Figure 2A). Thus, we compared the levels of mitochondrial proteins in total protein extracts prepared from hMIA40/Erv1 and hMIA40/b2 -hALR strains grown in the fermentable medium at lower (permissive conditions; Figure 3A) and higher (restrictive conditions; Figure 3B) temperature. The total protein analysis confirmed the moderate reduction of the levels of hMIA40 and equal 312
levels of other mitochondrial proteins in the hMIA40/Erv1 strain cultivated at the permissive temperature (Figure 3A). Importantly, when the cultures were shifted to restrictive 37◦ C, the levels of hMIA40 in the hMIA40/Erv1 cells were largely decreased compared with hMIA40/b2 hALR (Figure 3B). CCHL, which does not depend on MIA, and the proteins from other mitochondrial compartments were not affected, with the exception of Cox4, a subunit of cytochrome c oxidase in S. cerevisiae (Figure 3B). The effect on Cox4 likely reflects the instability of the cytochrome c oxidase reported in human cells defective in hALR (54), and is in agreement with the role of MIA in biogenesis of Cox17, a copper chaperone for cytochrome c oxidase (20). We investigated import of radiolabeled hMIA40 into mitochondria isolated from yeast strains grown in fermentable medium at the permissive temperature (Figure 4A). Under these conditions, the import of hMIA40 was less efficient in mitochondria with Erv1 compared to mitochondria with b2 -hALR (Figure 4A). This confirmed
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Figure 3: hALR is required to accumulate hMIA40 in the IMS. A and B) The amount of proteins in total protein extracts prepared from yeast strains is shown. Yeast cells were grown in YPS medium either at 19◦ C (permissive) (A) or the temperature was shifted to 37◦ C (restrictive) (B). Extracts were analyzed by reducing SDS-PAGE followed by Western blotting. WT, wild-type strain; *, nonspecific band.
the possibility that human sulfhydryl oxidase contributed to the biogenesis of hMIA40 by the involvement in its import. In line with total protein levels (Figure 3) and import (Figure 4A) analyses, the isolated mitochondria from the hMIA40/Erv1 and hMIA40/b2 -hALR cells grown at restrictive 37◦ C showed drastically reduced levels of hMIA40 (Figure 4B, lanes 1–4). Consequently, all the MIA pathway-dependent proteins were decreased in the mitochondria with hMIA40/Erv1 compared with the hMIA40/b2 -hALR mitochondria (Figure 4B, lanes 1–4). These changes were specific to the MIA pathway substrates because other mitochondrial proteins were unaffected with the exception of Cox4 (Figure 4B, lanes 5–8). Consistent with the steady-state protein analysis, the transport of radiolabeled Cox19, Cox17, Tim13, Tim9 and hMIA40 precursors into a proteaseprotected location (Figure S2A) and formation of the hMIA40-precursor conjugates was strongly inhibited in the hMIA40/Erv1 mitochondria (Figure S2B). We noticed that hMIA40 was present, albeit in smaller amounts, in total protein extracts (Figure 3B) but it was virtually absent in mitochondria prepared from the hMIA40/Erv1 strain (Figure 4B) grown under restrictive conditions. The inability of mitochondria with Erv1 to accumulate hMIA40 under restrictive conditions could lead to its mislocalization. We performed fractionation experiments of hMIA40/Erv1 and hMIA40/b2 -hALR strains (Figure 4C). The majority of hMIA40 was recovered in the mitochondrial fraction from the cells with b2 -hALR, even under restrictive conditions (Figure 4C, lanes 7–9,
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short exposure). When Erv1 was present, hMIA40 was not only decreased as expected based on previous experiments, but also partially (permissive conditions) or completely (restrictive conditions) mislocalized to the cytosolic fraction (Figure 4C, lanes 1–6, longer exposures). The distribution of mitochondrial proteins Tom70 and Mdj1 in the mitochondrial pellet and cytosolic proteins Pgk1 and Rpl17a in the post-mitochondrial supernatant showed that fractionation was successful (Figure 4C). Based on these results, we conclude that hMIA40 is properly targeted and efficiently accumulated in mitochondria only in the presence of hALR, suggesting a novel function of the human sulfhydryl oxidase in the biogenesis of hMIA40.
A disease-related form of hALR affects accumulation of hMIA40 in the IMS Di Fonzo et al. (54) found that a rare autosomal recessive myopathy connected with the development of cataract and respiratory-chain deficiency resulted from Arg194His substitution in hALR (54). To gain insights into the molecular basis of this disease, we used our yeast model. We obtained a yeast strain that harbors the mutated form of b2 -hALR (b2 -hALRR194H ) via plasmid shuffling according to the schema in Figure 1C. The strains that contained hMIA40 and either b2 -hALR or b2 -hALRR194H were spotted for growth analysis (Figure S3A). Compared with the strain that contained b2 -hALR, the growth of hMIA40/b2 -hALRR194H cells was only slightly impaired on the fermentable medium. We did not observe any differences in the steady-state levels of hMIA40 and other proteins in mitochondria isolated from the strains grown in the fermentable medium at the permissive temperature (Figure 5A). The only exception was the amount of hALR, which was approximately two-fold greater in hMIA40/b2 hALRR194H mitochondria than in mitochondria with b2 hALR (Figure 5A, lanes 1–4). The import of the IMS Cox19, Tim9 and Tim13 precursors into mitochondria and formation of hMIA40-precursor intermediates were unaffected under these conditions (Figure S3B,C). These results indicated that the Arg194His substitution had no effect on the function of hALR protein at the permissive temperature. Next, we monitored the growth of yeast strains in the fermentable medium after a shift to 37◦ C (Figure 5B). As expected, the wild-type strain grew the best, whereas the hMIA40/Erv1 and hMIA40/b2 -hALRR194H strains showed the highest sensitivity (Figure 5B). The analysis of total proteins indicated that the b2 -hALRR194H cells did not efficiently accumulate hMIA40 in contrast to b2 hALR, despite an increase in its amount (Figure 5C). We imported radiolabeled hMIA40 to mitochondria isolated from the permissive conditions that contained either b2 -hALR or b2 -hALRR194H and equal amounts of hMIA40. The import defect of hMIA40 in b2 -hALRR194H mitochondria compared to b2 -hALR mitochondria was observed (Figure 5D). Mitochondria isolated from the cells shifted to restrictive 37◦ C showed the reduction of hMIA40 in the b2 -hALRR194H mitochondria compared 313
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Figure 4: Mitochondrial import of hMIA40 is impaired in the absence of hALR. A) Import of radiolabeled hMIA40 precursor was performed into the mitochondria isolated from yeast strains grown in YPS medium at permissive 19◦ C. As a control, import reactions were blocked by 50 mM IAA. Samples were analyzed by reducing SDS-PAGE. B) The proteins levels in mitochondria isolated from yeast strains grown at restrictive 37◦ C were analyzed by reducing SDS-PAGE and Western blotting. C) Yeast strains grown in YPS medium at 19◦ C (permissive) or shifted to 37◦ C (restrictive) were subjected to cellular fractionation. Equal volumes of total (T), cytosolic (C) and mitochondrial (M) fractions were analyzed by reducing SDS-PAGE followed by Western blotting.
with mitochondria containing b2 -hALR (Figure 5E, lanes 1–4). Additionally, this effect was accompanied by a decrease in the IMS proteins that utilize the MIA pathway (Figure 5E, lanes 1–4). With the exception of Cox4, other mitochondrial marker proteins were present at similar levels in mitochondria with both hMIA40/b2 -hALRR194H and hMIA40/b2 -hALR (Figure 5E, lanes 5–8). The reduced levels of hMIA40 observed in the mitochondria with hMIA40/b2 -hALRR194H correlated well with the deficiency in import of the MIA precursors into mitochondria, oxidation of Tim13 and formation of the hMIA40 intermediate complexes (Figure S3D,E). Thus, under restrictive conditions, the presence of a diseaseassociated form of hALR, b2 -hALRR194H , impaired the accumulation of hMIA40 and other IMS proteins. The defect of b2 -hALRR194H in the mitochondrial accumulation of hMIA40 could result from the defective interaction between both proteins. To test this hypothesis, the strain containing His-tagged version of Mia40 (Mia40His ) was transformed with plasmids encoding b2 -hALR, b2 hALRR194H or Erv1 as a control. Isolated mitochondria were subjected to affinity purification via Ni-NTA (Figure S4A) as described previously (26,33). The efficiency of pull-down was assessed by the presence of Mia40His in the eluate. Both b2 -hALR and b2 -hALRR194H were also detected in eluate indicating the interaction between 314
human sulfhydryl oxidase and yeast Mia40His (Figure S4A, lanes 4 and 6). The efficiency of the interaction was difficult to interpret due to unequal amounts of b2 -hALR and b2 -hALRR194H in the load fractions (consistent with Figure 5). However, the comparison of b2 -hALR signals in the eluate versus load fraction gave an impression that b2 hALR interacted with Mia40 slightly more efficiently than b2 -hALRR194H (Figure S4A). Noncovalent interactions via disulfide bonds contribute to binding of Erv1 to Mia40 and these conjugates are visible on nonreducing SDS-PAGE (26,33,36,41,42,55,56). We used this strategy to look at the interactions between hMIA40 and b2 -hALR (Figure S4B). Using antibodies specific to hMIA40, we observed protein bands representing conjugates between hMIA40 and Erv1 or b2 -hALR that were identified by using yeast strain containing Erv1 and b2 -hMIA40 with higher molecular mass (due to inefficient b2 -presequence processing; 52). As a consequence i-b2 -hMIA40-Erv1 conjugate shows slower migration in comparison to hMIA40-Erv1 or hMIA40-b2 -hALR conjugate (Figure S4B, lanes 1–4). Using antibodies specific to hMIA40 and hALR, we observed that the formation of hMIA40-b2 -hALRR194H conjugates is slightly weaker compared to hMIA40-b2 -hALR complexes (Figure S4B, lanes 5–12). We conclude that the presence of Arg194His mutation in b2 -hALR may affect the stability of hMIA40-hALR complexes.
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Figure 5: The Arg194His substitution in hALR affects the biogenesis of hMIA40. A) Mitochondria were isolated from yeast grown in YPS medium at 19◦ C. The levels of mitochondrial proteins were analyzed by reducing SDS-PAGE followed by Western blotting. *, nonspecific band. B) Yeast were grown in YPS at restrictive 37◦ C. The density of cells was measured every hour at OD600 starting 30 min after the temperature was shifted to 37◦ C. SEM was calculated from four independent experiments. Filled circles, wild-type; filled triangles, hMIA40/b2 -hALR, open circles, hMIA40/Erv1; open triangles, hMIA40/b2 -hALRR194H . C) The total protein levels in the cells grown in YPS at 37◦ C are shown. Samples were analyzed by reducing SDS-PAGE followed by Western blotting. D) Radiolabeled hMIA40 precursor was imported into the mitochondria isolated from yeast strains grown in YPS at 19◦ C. As a control, import reaction was blocked by 50 mM IAA. Samples were analyzed by reducing SDS-PAGE. E) The protein levels in mitochondria isolated from yeast strains grown in YPS medium at 37◦ C are shown. Samples were analyzed by reducing SDS-PAGE and Western blotting.
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Sztolsztener et al.
A large pool of hMIA40 is found in the reduced state in yeast mitochondria The catalytic activity of Mia40 relies on the redox-sensitive disulfide bond formed by its CPC motif (see Figure 1A; 34, 39). The function assigned to the sulfhydryl oxidase Erv1 in the disulfide relay is the reoxidation of the CPC motif, subsequent to the release of substrate proteins in the oxidized state from Mia40 (25,38,42). Thus, we asked whether hALR deficiency is associated with changes in the hMIA40 redox state. To address this issue, we performed a thiol-trapping assay by incubating isolated mitochondria with iodoacetamide (IAA) or 4-acetamido4 -maleimidylstilbene-2,2 -disulfonic acid (AMS), alkylating agents that modify free sulfhydryl groups (Figure 6A). AMS, in contrast to IAA, increases the molecular mass of protein by 0.5 kDa. Thus, the reduced and oxidized forms of a protein can be distinguished by the difference in their mobility (33,38,41). To establish the thiol-trapping assay for human proteins, we used hMIA40/Erv1 mitochondria and mitochondria that contained the mutated form of hMIA40, hMIA40C4S to exclude the interference of an additional, nonconserved Cys4 residue (see Figure 1A). Incubation with AMS resulted in a change in the electrophoretic mobility of hMIA40, indicating the presence of free sulfhydryl groups that underwent modification by AMS (Figure 6A,B, lanes 1 versus 2). In the case of hMIA40C4S , the shifts of the protein bands were smaller because of a lack of Cys4 (Figure 6A,B, lane 2 versus 5). Applying mild reducing conditions resolves the CPC disulfide bond and liberates two thiols in the Mia40 molecule (25,38) (Figure 6A). We found that after dithiothreitol (DTT) treatment only a small pool of hMIA40 was shifted upward by AMS compared to the nontreated mitochondria (Figure 6B, lane 2 versus 3). This indicated that only a small fraction represented the form of hMIA40 (hMIA40oxi ) in which all of the Cys residues, including the CPC motif, were oxidized (except nonconserved Cys4). However, the majority of hMIA40 did not exhibit changes in mobility on the gel in response to DTT, indicating that this fraction of protein represents hMIA40 with the reduced CPC motif (hMIA40semi-red ; Figure 6B, lane 2 versus 3). Proper band assignment was verified using hMIA40C4S (Figure 6B, lane 5 versus 6). Thus, we established an assay that allowed us to address the redox state of hMIA40 in dependence on sulfhydryl oxidases. Mitochondria from the hMIA40/Erv1, hMIA40/b2 -hALR and hMIA40/b2 -hALRR194H strains, when isolated from yeast grown at the permissive temperature, contained detectable hMIA40 and therefore it was possible to subject these mitochondria to the thiol-trapping analysis. We did not observe any effect of the exchange of Erv1 for b2 -hALR/b2 -hALRR194H on the redox state of hMIA40 (Figure 6C, lanes 1–9). Moreover, the band pattern of hMIA40 did not change under restrictive conditions in the mitochondria with b2 -hALRR194H , despite reduced levels of hMIA40 (Figure 6C, lanes 10–15). Thus, the hMIA40 redox state was unaffected in the presence of b2 -hALRR194H even under the restrictive conditions. Collectively, we demonstrated that hMIA40 exists in 316
two different redox forms, the most abundant of which has the CPC motif reduced. The presence of Erv1, b2 hALR or b2 -hALRR194H did not lead to the change in the redox state of hMIA40 accumulated in mitochondria. Erv1, in addition to reoxidation of CPC motif, was recently shown to interact with the CX9 C motif of Mia40 (56). Thus, it is tempting to speculate that the defect in this interaction contributes to the defect observed for hMIA40/Erv1 and hMIA40/b2 -hALRR194H leading to the deficient mitochondrial accumulation of hMIA40.
Disease-related change in Erv1 affects accumulation of yeast soluble Mia40 in the IMS We hypothesized whether the novel function of hALR in the biogenesis of hMIA40 is also relevant to its yeast homologues. We constructed the strain harboring the mutant form of Erv1 by introducing the substitution of conserved Arg182 in Erv1 (R182H) that mimics the disease-causing change R194H in hALR (54). This strain and strains producing b2 -hALR or b2 -hALRR194H were transformed with the plasmid coding for the conserved C-terminal domain of Mia40 (Mia40core ). In contrast to wild-type Mia40, Mia40core utilizes the MIA pathway and is soluble in the IMS (52). The resulting strains contained wild-type membrane-anchored Mia40, Mia40core and various versions of Erv1/hALR. We analyzed the presence of Mia40 and Mia40core in isolated mitochondria and in total protein extracts from cells grown at restrictive conditions (Figure 7). We observed that the levels of Mia40core significantly decreased in mitochondria (Figure 7A) and in total protein extracts (Figure 7B) derived from the cells containing mutant forms of sulfhydryl oxidases. This was not the case for native membrane anchored Mia40. Also, the control mitochondrial proteins were not affected (Figure 7A,B). Thus, soluble Mia40core was not efficiently accumulated in mitochondria in the presence of the disease-related forms of sulfhydryl oxidases.
Discussion The MIA represents a unique protein import and sorting pathway involved in mitochondrial biogenesis, because it functionally couples the translocation of a subset of precursor proteins into the mitochondrial IMS with their oxidative folding. As a net result, proteins stabilized by disulfide bonds are accumulated in the mitochondrial IMS in their mature form (15–19). The key constituents of the MIA import system, Mia40 and Erv1, are conserved among the majority of eukaryotic organisms. Detailed knowledge about the molecular basis of MIA function was obtained in studies performed on the yeast S. cerevisiae. However, whether and by what mechanisms the eukaryotic MIA components operate in the process of the import and oxidation of IMS proteins remained elusive. We took advantage of yeast as a model system that has proven useful for understanding the biogenesis of mitochondrial proteins and its dysfunction
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Mitochondrial Protein Folding and Translocation A
Figure 6: hMIA40 is partially reduced in yeast mitochondria. A) Schematic representation of thiol-trapping experiment showing hMIA40 and hMIA40C4S redox state upon modification of cysteine residues with AMS. B and C) Mitochondria were isolated from yeast strains grown under conditions as indicated. In brackets the number of AMS molecules attached to hMIA40 or hMIA40C4S free thiol groups is shown. oxi , oxidized; red , reduced; semi-red , partially reduced.
(57–60). By expressing hMIA40 and hALR in yeast that lack the endogenous Mia40 and Erv1 proteins, we demonstrated that hMIA40 and hALR complement a lifeessential function of Mia40 and Erv1 in the biogenesis of IMS proteins. Several features of Mia40 have been recapitulated in our yeast model for the human MIA disulfide relay pathway, including the import of IMS precursors and formation of disulfide-bonded Mia40 intermediate complexes followed by oxidation of IMS client proteins. We also found important variations in the function of the human MIA pathway compared with its yeast counterparts. Surprisingly, hMIA40 was found in significant amounts in a form in which the redox-active CPC motif responsible for IMS proteins oxidation is reduced. However, our results showed that hMIA40 effectively acted in the biogenesis of IMS proteins despite having partially reduced CPC motif. Our findings are supported by a previous study, in which hMIA40 was found to exist in different redox states in human cells (43). Compelling evidence provided by studies in yeast and with recombinant proteins assigned a role for the sulfhydryl oxidase Erv1 in the reoxidation of the Mia40 CPC motif after completion of the substrate’s oxidative folding (25,38,40–42). Interestingly, in our yeast model of the human MIA pathway, this sulfhydryl oxidase-dependent step was not limiting. A deficiency in sulfhydryl oxidase activity caused by either the presence of yeast Erv1 (obviously, Erv1 is not a perfect
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match for hMIA40) or amino acid substitution in hALR, did not decrease the amount of fully oxidized hMIA40. Rather, a shortage in sulfhydryl oxidase decreased the overall levels of hMIA40 in mitochondria and total cells. Interestingly this phenotype is fully recapitulated in the case of soluble version of yeast Mia40 (Mia40core ). Thus, the role of sulfhydryl oxidases in determining the productive accumulation of soluble version of hMIA40 described in our study is evolutionarily conserved. However, this function is not critical in lower eukaryotes such as yeast, in which Mia40 is transported via the TIM23 pathway independently on its oxidative folding and remains bound to the inner mitochondrial membrane (22,43,52). This is in contrast to human (and other higher Eukaryotes), where dysfunction in the ability of hALR to regulate the biogenesis of hMIA40 results in pathology (54). Also, our results provide some mechanistic insights into the role of sulfhydryl oxidase in accumulation of hMIA40. In addition to the CPC motif, Mia40 harbors two other disulfide bonds formed by the conserved twin CX9 C, for which a structural role was assigned (see Figure 1A; 39). The mitochondrial localization of hMIA40 depends on its oxidative folding, i.e. formation of structural disulfide bonds within the CX9 C motifs, because removal of these cysteine residues in hMIA40 causes the defective import of hMIA40 into mitochondria (43). The sulfhydryl oxidase is involved in the import-coupled oxidative folding of hMIA40 (and the Mia40 proteins from different organisms 317
Sztolsztener et al. A
pFL39-b2 -(1–84)-hALR (pGB9550) or pFL39-b2 -(1–84)-hALRR194H -TRP1 (pMS105) plasmid. Yeast strains containing wild-type Mia40 and pFL39derived plasmids with ERV1 (pGB9011), ERV1R182H (pMS106), b2 -(1–84)hALR (pGB9550) or b2 -(1–84)-hALR R194H (pMS105) were transformed with pRS416 derived plasmid with MIA40core (pKO1 = pRS416-MIA40P MIA40core -MIA40T -URA3 ).
Growth conditions and isolation of mitochondria
Figure 7: Erv1 is required for accumulation of yeast IMSsoluble Mia40. A and B) Yeast were grown in YPS medium at 37◦ C. The protein levels in isolated mitochondria (A) or in total protein extracts (B) are shown. Samples were analyzed by reducing SDS-PAGE followed by Western blotting. *, nonspecific bands.
that are not membrane-anchored) that likely relies on a direct interaction between Mia40 and sulfhydryl oxidase. Indeed, Erv1 was recently shown to interact with Mia40 in a manner that depends on the cysteine residues of twin CX9 C motif (56). Altogether, we suggest a novel function of sulfhydryl oxidase in the oxidation of cysteine residues in the twin CX9 C motif of Mia40 that is required to efficiently accumulate soluble Mia40 in the IMS of mitochondria. Interestingly, Mia40 that cannot undergo the sulfhydryl oxidase-driven biogenesis in mitochondria is only partially recovered in the cytosol suggesting the involvement of the protein clearance mechanisms that remove mislocalized and unfolded hMIA40.
Materials and Methods Yeast strains and plasmids The S. cerevisiae strains used in this study are derivatives of YPH499 (MATa, ade2-101, his3-200, leu2-1, ura3-52, trp1-63, lys2-801) (61) and are listed in Table S1. Genetic manipulations and plasmid shuffling were performed according to standard procedures. The plasmids encoding b2 hALRR194H , Erv1R182H and hMIA40C4S were generated by site-directed mutagenesis using pFL39-b2 -(1–84)-hALR-TRP1 (pGB9550), pFL39-ERV1TRP1 (pGB9011) and pFL39-hMIA40-TRP1 (pGB9347) plasmids as templates. Mia40His strain was transformed with pFL39-ERV1 (pGB9011),
Yeast strains were grown in liquid or on solid [2.5% (w/v) agar] respiratory YPG or fermentative YPS medium containing 1% (w/v) yeast extract, 2% (w/v) bacto-peptone with 3% (w/v) glycerol or 2% (w/v) sucrose as a carbon source. Sucrose was chosen as a fermentative carbon source. The selective medium (0.67% yeast nitrogen base, 2% glucose supplemented with appropriate amino acids and nucleotides) was used for the selection of transformants. To obtain mitochondria by differential centrifugation (62) the yeast strains were grown at 19◦ C for 24 h or after 16 h of growth at 19◦ C the temperature was shifted to 37◦ C for 4–8 h. The concentration of mitochondria was adjusted to 10 mg of protein/mL in SM buffer (250 mM sucrose, 10 mM MOPS/KOH, pH 7.2). For cellular fractionation 40 OD600 units of yeast cells were lysed in homogenization buffer [0.6 M sorbitol, 10 mM Tris–HCl pH 7.4, 2 mM phenylmethylsulfonyl fluoride (PMSF)]. After pelleting cell debris and nuclei (3000× g , 5 min), a portion was withdrawn as a total fraction (T) and the remaining solution was subjected to centrifugation (20 000× g , 10 min) resulting in mitochondrial pellet (P) and cytosolic (C) fractions. Fractions T and C were precipitated with 10% trichloroacetic acid, and washed with ice-cold acetone. Samples were denatured in the Laemmli buffer. The amounts representing equal volumes for each fraction were analyzed by reducing SDS-PAGE followed by Western blotting. To generate mitoplasts, isolated mitochondria were subjected to hypoosmotic swelling in a buffer containing 1 mM EDTA and 10 mM MOPS/KOH, pH 7.2 (63). For control, mitochondria were incubated in isoosmotic buffer with 250 mM sucrose. After centrifugation (20 000× g , 15 min, 4◦ C) the post-mitochondrial supernatants were precipitated with 10% trichloroacetic acid, washed with ice-cold acetone and solubilized in Laemmli buffer.
In vitro import of precursor proteins Radiolabeled precursor proteins were synthesized with the TNT SP6 coupled transcription/translation kit (Promega) or reticulocyte lysates (Promega) in the presence of [35 S]methionine (GE Healthcare). Precursors of Cox and small Tim proteins were denatured in urea buffer (8 M urea, 30 mM MOPS pH 7.2, 10 mM DTT) (29). hMIA40 was synthesized in the presence of 10 mM DTT. The import of radiolabeled precursors into isolated yeast mitochondria was performed at 30◦ C in import buffer (250 mM sucrose, 5 mM MgCl2 , 80 mM KCl, 10 mM MOPS/KOH, 5 mM methionine, 10 mM KH2 PO4 , pH 7.2). Import was stopped by placing samples on ice in the presence of 50 mM IAA (Sigma-Aldrich) followed by the treatment with 50 μg/mL proteinase K for 15 min to remove nonimported protein excess. The activity of proteinase K was inhibited by the addition of 2 mM PMSF. After wash, mitochondrial pellet was resuspended in Laemmli buffer containing either 50 mM IAA (nonreducing) or 50 mM DTT (reducing) and analyzed by SDS-PAGE. Radiolabeled proteins detected by digital autoradiography (Phosphorimager Storm 820; Amersham Bioscience) and analyzed by IMAGEQUANT 5.0 software (Molecular Dynamics).
Thiol-trapping experiments Thiol modifications were performed essentially as described previously (33,38). Isolated mitochondria were preincubated at 20◦ C in SM buffer (250 mM sucrose, 10 mM MOPS/KOH, pH 7.2) with 50 mM DTT for 30 min. Mitochondrial pellets were resuspended in Laemmli buffer containing either 50 mM IAA or 15 mM AMS (Invitrogen, Life Technologies). Samples were analyzed by nonreducing SDS-PAGE and Western blotting.
Miscellaneous Total protein extracts were prepared by alkaline lysis (64). Protein concentration was determined using Roti-Quant reagent (Carl Roth GmbH).
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Mitochondrial Protein Folding and Translocation Proteins were separated by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore). In some cases Western blots were scanned with an ImageQuant LAS4010 system (GE Healthcare). Images were processed using Adobe Photoshop CS4.
Acknowledgments We thank Kamila Ornoch for the pKO1 plasmid. We are grateful to Drs ¨ Nikolaus Pfanner, Chris Meisinger, F.-Nora Vogtle for discussion. This work was supported by the grant from Ministry of Science and Higher Education in Poland (NN301 298337), the Welcome Programme (Foundation for Polish Science) co-financed by the EU within the European Regional Development Fund, and the EMBO Installation grant. The authors declare that they have no conflict of interest.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1: Saccharomyces cerevisiae strains used in this study. Figure S1: Mitochondria with hMIA40 import IMS precursor proteins. Mitochondria were isolated from yeast strains grown at 19◦ C in the YPG medium. A) The levels of proteins were assessed by reducing SDSPAGE followed by Western blotting. B and C) Isolated mitochondria were subjected to the import assays by incubation with radiolabeled Cox19, Cox17, Tim13 and Tim9 precursors. As a control, import reactions were blocked by 50 mM iodoacetamide (IAA). Samples were analyzed by reducing (B) or nonreducing (C) SDS-PAGE followed by autoradiography. oxi , oxidized; red , reduced; WT, wild-type strain; *, nonspecific bands. Figure S2: Import of the MIA-dependent proteins is impaired in the presence of Erv1 under restrictive conditions. A and B) Import of radiolabeled Cox19, Cox17, Tim13, Tim9 and hMIA40 precursors was performed into mitochondria isolated from yeast strains grown in YPS medium at 37◦ C. As a control, import reactions were blocked by 50 mM IAA. Samples were analyzed by reducing (A) or nonreducing (B) SDS-PAGE followed by autoradiography. *, nonspecific bands. Figure S3: Mutant version of hALR impairs the function of the MIA pathway under restrictive conditions. A) Yeast strains were spotted onto YPG and YPS plates in serial 10-fold dilutions. B–E) The radiolabeled precursors of Cox19, Tim13 and Tim9 proteins were imported into mitochondria isolated from yeast strains grown in YPS medium at 19◦ C (B, C) or at 37◦ C (D, E). As a control, import reactions were blocked by 50 mM IAA. Samples were analyzed by reducing (B, D) or nonreducing (C, E) SDS-PAGE and autoradiography. oxi , oxidized; red , reduced. Figure S4: Analysis of hMIA40-hALR interactions. A) The Mia40His strains with plasmids carrying ERV1, b2 -hALR or b2 -hALRR194H were subjected to mitochondrial isolation. Mitochondria were solubilized in digitonin-containing buffer and Mia40His complexes were isolated via affinity purification. Load (3%) and eluate (100%) fractions were separated by reducing SDS-PAGE followed by Western blotting. B) Identification of conjugates formed between hMIA40/b2 -hMIA40 and Erv1/b2 -hALR/b2 hALRR194H in mitochondria isolated from yeast grown at 19◦ C. Samples were analyzed by nonreducing SDS-PAGE followed by immunodecoration with anti-hMIA and anti-hALR antibodies. WT, wild-type strain.
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