Human Molecular Genetics, 2001, Vol. 10, No. 3 259-269
© 2001 Oxford University Press
Distinct roles for two N-terminal cleaved domains in mitochondrial import of the yeast frataxin homolog, Yfh1p
1Department of Physiology, University of Pennsylvania School of Medicine, D403 Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104-6085, USA and 2Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104-6100, USA
Received 23 October 2000; Revised and Accepted 1 December 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The yeast frataxin homolog (Yfh1p) participates in mitochondrial iron homeostasis. The phenotypic defects of the
yfh1 mutant include drastic accumulation of iron in mitochondria and slow growth. The Yfh1p precursor protein contains two N-terminal domains that are sequentially cleaved by the matrix processing peptidase on import into mitochondria, generating the mature protein. We have precisely mapped these two cleavage sites. Mutations blocking the first or the second cleavage of Yfh1p do not interfere with its in vitro import or with its ability to complement phenotypes of the yfh1 mutant strain. Distinct roles have been ascertained for the two cleaved domains of Yfh1p. The first cleaved domain (domain I) is sufficient for in vitro mitochondrial import of a non-mitochondrial passenger protein. However, neither domain I nor other matrix-targeting signals alone can support efficient in vitro import of mature Yfh1p. The second cleaved domain (domain II) is required as a spacer between a targeting signal and mature Yfh1p. Likewise, when Yfh1p constructs lacking domain I or II are expressed in vivo, they fail to attain appreciable steady-state amounts in mitochondria and cannot complement phenotypes of the yfh1 mutant. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Iron is indispensable for most forms of life because of its role as a cofactor for many essential proteins (1). In eukaryotic cells, many of these proteins reside within mitochondria and contain iron either as Fe–S or heme. Conversely, iron can be toxic through its participation in reactions that generate free radicals and iron overload states have been implicated in hemochromatosis (2), sideroblastic anemia (3), immune disorders (4) and neurodegenerative diseases (5). The requirement for iron and its toxicity has therefore dictated the need for mechanisms to regulate cellular and organelle iron and iron protein levels.
The protein Yfh1p is involved in iron homeostasis in yeast and is highly homologous to human frataxin, a protein deficient in patients with Friedreich ataxia (FRDA). FRDA is an autosomal recessive disorder and represents the most common inherited ataxia (6). Although the pathogenesis of FRDA remains unclear, the cellular phenotype of the yfh1 yeast mutant is remarkably similar to that observed in cells of affected tissues from patients with FRDA. The yfh1 yeast mutant accumulates iron within mitochondria and is deficient in mitochondrial iron proteins such as aconitase and some respiratory complexes (7–9). Likewise, cardiac muscle from patients with FRDA contains excess iron and lacks activities of aconitase and of respiratory complexes (10). Iron accumulation, however, may not be the only defect in FRDA patients, as suggested by a recent study of frataxin knockout mice which showed early embryonic lethality without iron accumulation (11). Nevertheless, the human frataxin is able to complement many of the phenotypes of the yfh1 yeast mutant (12,13), suggesting that the human protein exerts its effects via a pathway similar to that in yeast.
The Yfh1p precursor protein is nuclear encoded, translated on cytosolic ribosomes and imported into mitochondria. On import, two domains are removed from the N-terminus by an unusual two-step maturation process (14). An initial proteolytic cleavage removes a region of 
2 kDa (domain I), generating an intermediate fragment. The intermediate is then further processed by a cleavage that removes an additional 4 kDa (domain II), generating the final mature form of Yfh1p. We (14) and others (15) have noted that in a yeast mutant lacking a low-abundance mitochondrial Hsp70 chaperone, Ssq1p, conversion of the intermediate to the mature form is kinetically much slower compared with the wild-type mitochondria. Interestingly, the phenotypes of ssq1 and yfh1 mutants are very similar (7,8,14,16): both exhibit increased cellular iron uptake and the mitochondria dramatically accumulate much of this iron. These earlier studies raise the question of the role of Yfh1p processing in iron homeostasis. We have now determined the exact boundaries of the cleaved domains I and II of the Yfh1p precursor protein. This has allowed us to examine the roles of the two cleaved domains of the Yfh1p signal sequence and the effect of non-processing of these domains on Yfh1p functions. Here we show that proteolytic removal of domain II was not required for import or complementation of iron regulatory phenotypes of the yfh1 mutant. However, each of the two cleaved domains was required for efficient import of Yfh1p and was found to play a distinct role in this process. Domain I served as a matrix-targeting signal and could be replaced by other similar signals. Domain II functioned as a spacer and could be replaced by an unrelated sequence.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mapping the cleavage sites involved in maturation of Yfh1p
We demonstrated previously that on import the N-terminus of the Yfh1p precursor (29 kDa) was processed in two steps (14). An initial cleavage removed
2 kDa from the precursor protein, generating an intermediate fragment (i) and a subsequent cleavage removed an additional 4 kDa from the latter to generate the mature form (m). The processed forms did not appear if valinomycin was present in the import reactions, indicating that a membrane potential was required for import to proceed. Both intermediate and mature molecules were protected from digestion by exogenously added trypsin and sedimented with mitochondria on centrifugation. In ssq1-4 mutant mitochondria, the import and the first processing step were normal but the second processing step was specifically impaired (Fig. 1A) (14).
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To further evaluate Yfh1p processing, we examined the cleavage pattern generated by bacterially expressed and purified matrix processing peptidase (MPP) in the absence of mitochondria (Fig. 1A, lane 2). Two additional bands were generated at the expense of the precursor protein. More importantly, the MPP-cleaved products were identical in size to the intermediate and mature forms of Yfh1p generated during its maturation within mitochondria. These results indicate that the maturation of Yfh1p is mediated by MPP alone, as has been suggested (17,18).
To precisely map the MPP cleavage sites in the Yfh1p precursor, we took advantage of the import pattern of a chimeric protein, Yfh1p–Protein A, where Protein A is linked in-frame to the C-terminus of the Yfh1p precursor. As expected, on import this chimeric protein was also processed in two steps at the N-terminus and both the intermediate and the mature forms retained the Protein A moiety at their C-termini (14). The chimeric precursor protein with a C-terminal His6 tag was overexpressed in bacteria, purified on Ni-NTA resin and then used in a large-scale import assay. Following trypsin treatment to remove unimported Yfh1p–Protein A, mitochondria were solubilized with Triton X-100 and chromatographed on rabbit IgG–Sepharose. As the import reaction was performed with wild-type mitochondria, the affinity eluate contained mostly the mature form and very little of the intermediate form (data not shown). In a separate experiment, the chimeric precursor was treated with purified MPP in the absence of mitochondria and the reaction mixture was subjected to IgG–Sepharose chromatography. Under these conditions, some of the precursor molecules were converted to the intermediate form and very little of the mature form was detected (data not shown). It should be noted that in the absence of mitochondria, the conversion of iYfh1p to mYfh1p by purified MPP was less efficient and required higher concentrations of purified MPP and longer incubation times (Fig. 1A) (18).
The bands corresponding to the intermediate and the mature forms of chimeric Yfh1p were excised and subjected to N-terminal protein sequencing. The N-terminal sequence of iYfh1p was Met-Ile-Ala-Ala-Ala, corresponding to residues 21–25 of the Yfh1p precursor (Fig. 1B). The first cleavage of the Yfh1p precursor by MPP must therefore occur between residues 20 and 21. The amino acid sequence preceding the cleavage (17GRR20Y^21MIAA25A) matches a consensus for MPP cleavage seen in many precursor proteins, i.e. an arginine at position –2, and often also at position –3, with respect to the cleavage site (in bold) (19). Earlier studies (18,20) predicted the first cleavage site by comparing the mobilities of iYfh1p with various N-terminally truncated forms of Yfh1p; amino acid sequencing of purified iYfh1p was not performed to confirm the prediction. One study (20) predicted this site correctly and another study (18) predicted the first cleavage site between residues 21 and 22 [as opposed to residues 20 and 21 (this study)].
The N-terminal sequence of mYfh1p was determined to be Val-Glu-Ser-Ser-Thr, corresponding to residues 52–56 of the Yfh1p precursor (Fig. 1B). The second cleavage during maturation of Yfh1p must therefore occur between residues 51 and 52, in agreement with recent studies (20,21). This second cleavage site, 48QKR51F^52VESS56T, also fits the consensus for an MPP cleavage site although it has a K (instead of R) at position –3. For brevity, residues 1–20 and 21–51 will be referred to as domains I and II, respectively.
MPP cleavage of domains I and II are independent of each other
The cleavage mediated by MPP often requires basic residues at positions –2 and –3 from the cleavage site (19). To investigate whether the second MPP cleavage depends on the first MPP cleavage, arginine residues at positions 18 and 19 of the Yfh1p precursor were mutated to glycine residues. As expected, no intermediate fragment was detected when Yfh1p(R18G, R19G) was incubated with purified MPP in the absence of mitochondria or on its import into mitochondria (Fig. 2A). The band indicated by an asterisk represents a presumed internal initiation product from the translation reaction and is not a processing intermediate. Interestingly, however, MPP cleavage at the second site remained unaffected, thereby directly generating mYfh1p (Fig. 2A, lanes 2–4).
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To investigate the effects of non-processing of domain II on Yfh1p functions, we constructed another mutant, Yfh1p(K49G, R50G). This mutant was efficiently imported into isolated wild-type mitochondria and processed to generate iYfh1p (Fig. 2B). The second processing step (i.e. the removal of domain II) was completely blocked and no mYfh1p was generated (lanes 3–6). Likewise, when the mutant Yfh1p was incubated with purified MPP, only iYfh1p was generated (lane 2).
These findings were further confirmed by in vivo experiments. We tested the ability of various Yfh1p constructs expressed in vivo to complement the yfh1 mutant. Wild-type Yfh1p and vector alone served as positive and negative controls, respectively. The constructs encoding various precursor proteins were placed under control of the native YFH1 promoter and integrated into the genome of the yfh1 shuffle strain. The covering YFH1 plasmid was ejected by exposure to cycloheximide. Mitochondrial levels of Yfh1p were evaluated by western blots of isolated mitochondria using monospecific polyclonal antibodies against the Yfh1p precursor. Almost identical levels of two unrelated proteins, Put2p and Por1p, served as internal controls. When Yfh1p(R18G, R19G) was expressed in the yfh1 shuffle strain, no defect in the second processing step was observed and the level of mYfh1p in isolated mitochondria was comparable to that of the wild-type. On the other hand, the mutant Yfh1p(K49G, R50G) was detected only as the intermediate form with uncleaved domain II (Fig. 2C, top). Note that although marker proteins were detected at a comparable level, the Yfh1p(K49G, R50G) intermediate showed a stronger signal than the mature form present in wild-type Yfh1p or Yfh1p(R18G, R19G). This may indicate an increased level of the intermediate or an increased antibody binding, as anti-Yfh1p antibodies were raised against the entire precursor protein.
Complementation of the yfh1 phenotypes was evaluated with respect to cell growth (Fig. 3A) and cellular iron uptake (Fig. 3B). The yfh1 mutant grows slowly and exhibits derepressed cellular iron uptake. These phenotypes resemble the ssq1-4 mutant phenotypes (14). The mutant Yfh1p(K49G, R50G) was able to efficiently complement the yfh1 phenotypes. Thus, non-processing of domain II does not interfere with Yfh1p functions. In addition, these results show that the impaired processing of Yfh1p domain II that occurs in ssq1 mutant mitochondria cannot account for the mutant phenotypes of the ssq1-4 strain.
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Domain I serves as a typical matrix-targeting signal
To determine the roles of domains I and II of the Yfh1p signal sequence, we made several Yfh1p chimeric constructs (Fig. 4). Note that efficient cleavage of precursor proteins by MPP often requires as few as two consensus N-terminal amino acids from the mature product (22); constructs were designed accordingly to permit MPP cleavage. These constructs were evaluated in assays for in vitro import, in vivo mitochondrial protein levels and complementation of the
yfh1 mutant. A summary of these results is shown in Figure 4.
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In vitro import reactions were performed using 35S-labeled precursor proteins synthesized in rabbit reticulocyte lysate. Following import, untreated and trypsin-treated samples were analyzed. The intermediate and/or mature products that were generated as a result of import and maturation within mitochondria were then compared with the cleavage products generated when precursor proteins were treated with purified MPP in the absence of mitochondria. Results can be summarized as follows. (i) When residues 1–53 of the Yfh1p precursor were linked to Protein A, the resulting construct (Yfh1p1–53–Protein A, 38 kDa) was imported and processed (Fig. 5A). Note that import reactions were carried out with wild-type mitochondria at 30°C for 30 min. Under this longer incubation at a higher temperature, the intermediate fragment could not be detected; only the final mature product was detected. However, processing by MPP in the absence of mitochondria was slower and both intermediate and mature forms could be detected. (ii) When residues 1–22 of the Yfh1p precursor were linked to Protein A, the resulting construct (Yfh1p1–22–Protein A, 34 kDa) was also imported and processed to a form which remained resistant to external trypsin attack (Fig. 5B). (iii) When domain I was deleted from the Yfh1p precursor leaving domain II intact, the resulting construct
20Yfh1p (27 kDa) failed to be imported (Fig. 5C). However, as expected, 20Yfh1p was processed to mYfh1p in one step by purified MPP. Likewise, when residues 21–53 of the Yfh1p precursor were linked to Protein A, the resulting construct (Yfh1p21–53–Protein A, 36 kDa) failed to be imported although it was processed by purified MPP (Fig. 5D). Together, these results show that domain I of the Yfh1p precursor contains necessary and sufficient information for mediating mitochondrial import of a passenger protein (Protein A). In contrast, domain II by itself is not sufficient for import of mYfh1p or Protein A.
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We then investigated the import of two other chimeric proteins, Cox4p1–22–iYfh1p (30 kDa) (Fig. 5E) and Put2p1–17–iYfh1p (30 kDa) (Fig. 5F). In the former, domain I of Yfh1p was replaced by the first 22 amino acids of the Cox4p precursor protein with a predicted MPP cleavage site between residues 17 and 18. In the latter, domain I of Yfh1p was replaced by the first 17 amino acids of the Put2p precursor with a predicted MPP cleavage site between residues 15 and 16. Both these chimeric proteins are therefore expected to have two MPP cleavage sites. Indeed, these chimeric proteins, like the authentic Yfh1p, were efficiently imported into mitochondria and were processed in two steps by purified MPP. Thus, domain I could be functionally replaced by other known mitochondrial signal sequences. These results also validate the experiments described in the following sections using chimeric proteins containing Put2p or Cox4p signal sequences in order to determine the role of domain II in Yfh1p import and functions.
Domain II performs a critical function separating the targeting signal from mYfh1p
To investigate the role of domain II of the Yfh1p signal sequence in import, it was deleted from the authentic Yfh1p. Surprisingly, the resulting construct, Yfh1p1–22–mYfh1p (25 kDa), with intact domain I followed by the mature Yfh1p, failed to be imported to any appreciable extent (Fig. 6A), suggesting that domain I alone is not sufficient for efficient import of mYfh1p. We then tested whether Cox4p or Put2p signal sequences, which were capable of mediating efficient import of iYfh1p (with intact domain II), would mediate import of mYfh1p (lacking domain II). Like Yfh1p1–22–mYfh1p, the chimeric proteins Cox4p1–22–mYfh1p (26 kDa) (Fig. 6B) and Put2p1–17–mYfh1p (26 kDa) (Fig. 6C) also failed to be efficiently imported into isolated mitochondria. These results suggest that in the absence of domain II, mitochondrial targeting signals cannot mediate import of mYfh1p.
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Precursor proteins must be at least partially unfolded prior to import through protein-conducting channels of a limited diameter at the outer mitochondrial membrane (for reviews see refs 19 and 23). Lack of import of precursors could be due to their failure to be properly unfolded outside mitochondria. To investigate this possibility, we took advantage of our earlier observations that bacterially expressed and urea-denatured Yfh1p as well as Put2p can be efficiently imported into mitochondria (14,24). Three precursor proteins, Yfh1p1–22–mYfh1p, Put2p1–17–mYfh1p and Put2p1–17–iYfh1p, each with a C-terminal His6 tag, were expressed in bacteria, purified to homogeneity on Ni-NTA agarose in the presence of 8 M urea (data not shown) and tested for import into mitochondria. Again, no significant import was observed with Yfh1p1–22–mYfh1p (data not shown) or Put2p1–17–mYfh1p (Fig. 6D). Thus, prior denaturation cannot obviate the import defects of these proteins lacking domain II of Yfh1p. On the other hand, urea-denatured Put2p1–17–iYfh1p was efficiently imported and served as a positive control (Fig. 6E). These results suggest that the mitochondrial targeting signal must be separated from the mYfh1p moiety and domain II therefore acts as a spacer between these two domains facilitating import.
To evaluate the specificity of the spacer function of domain II, we tested import of two other constructs, Put2p1–32–m*Yfh1p (28 kDa) (Fig. 7A) and Put2p1–32–mYfh1p (28 kDa) (Fig. 7B). The former contains the authentic second cleavage site from Yfh1p, whereas the latter does not. In both cases, a longer N-terminal fragment of the Put2p precursor was attached to mYfh1p to investigate whether a spacer (24 amino acids for Put2p1–32–m*Yfh1p or 19 amino acids for Put2p1–32–mYfh1p) separating the targeting signal from mYfh1p would restore import. Note that Put2p1–17–iYfh1p, Put2p1–32–m*Yfh1p and Put2p1–32–mYfh1p are almost identical except that, in the latter two, domain II is replaced by unrelated Put2p protein sequences. Indeed, the latter two proteins were imported as efficiently as Put2p1–17–iYfh1p. As expected, Put2p1–32–m*Yfh1p was processed in two steps whereas Put2p1–32–mYfh1p was processed in one step by MPP. Thus, the spacer function is not specific for domain II and can be achieved by other unrelated sequences.
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To investigate the requirement of domain II for efficient Yfh1p import in vivo, we tested the complementing ability of various Yfh1p constructs expressed under control of the native YFH1 promoter in a
yfh1 shuffle strain (Fig. 7C). Wild-type Yfh1p and vector alone served as positive and negative controls, respectively. Almost identical levels of two unrelated mitochondrial proteins, Tom40p and Put2p, served as internal controls. As expected, wild-type Yfh1p and Put2p1–17–iYfh1p were detected in mitochondria at comparable levels and were able to completely complement the slow growth and iron accumulation phenotypes of the yfh1 mutant. On the other hand, Yfh1p was not detected in mitochondria isolated from cells expressing 20Yfh1p and complementation was not achieved (Fig. 4). These results confirm that Yfh1p must be imported into mitochondria for its function, and that domain I of Yfh1p is required for mitochondrial import. Constructs lacking domain II, Yfh1p1–22–mYfh1p (Fig. 7C) or Put2p1–17–mYfh1p (Fig. 4), failed to mediate any appreciable complementation and Yfh1p levels within mitochondria were only 10–15% compared with the wild-type (Fig. 7C). Thus, domain II is required for Yfh1p import both in vitro and in vivo. The minimum level of Yfh1p that is required for complementation of the yfh1 mutant remains to be determined. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The yeast frataxin homolog (Yfh1p) is a nuclear-encoded mitochondrial protein that participates in cellular and mitochondrial iron homeostasis. The yeast protein is homologous to a human protein implicated in the neurodegenerative disease FRDA, and evidence exists that the human protein also functions in iron homeostasis of mitochondria. Here we have examined import of the Yfh1p precursor protein into mitochondria and the role of two cleaved N-terminal domains in this process.
On import into mitochondria, the Yfh1p precursor was processed in two steps entirely by the MPP and we have precisely mapped these cleavage sites. The first processing step removed 20 amino acids from the N-terminus of the precursor (domain I), generating an intermediate form of Yfh1p. This intermediate was then processed again by MPP and an additional 31 N-terminal amino acids (domain II) were removed, generating the final mature form of Yfh1p. The processing of Yfh1p in two steps entirely by MPP is quite unusual. The signal sequence of most precursor proteins targeted to the mitochondrial matrix is removed in one step by MPP. However, a subset of precursors (e.g. the Rieske Fe/S protein encoded by RIP1) undergoes a second processing step: the precursor is first cleaved by MPP to generate an intermediate which is subsequently processed to the mature form by another enzyme, mitochondrial intermediate peptidase (MIP, encoded by OCT1) (25,26). Prior MPP cleavage is essential for MIP cleavage to occur. For example, when basic amino acids adjacent to the MPP cleavage site are mutated, both MPP and MIP cleavages are completely blocked (27). Interestingly, analogous mutations at positions 18 and 19 of the Yfh1p precursor completely blocked the first MPP processing step but did not significantly interfere with the second processing step. Likewise, similar mutations at positions 49 and 50 of the Yfh1p precursor completely blocked the second processing step leaving the first processing step unaffected. The two processing steps in Yfh1p are therefore independent of each other.
The delayed second processing step of Yfh1p that occurs in ssq1 mutants lacking the low-abundance Hsp70 chaperone was noted by us (14) and also recently by others (15). Interestingly, mutants lacking Ssq1p themselves exhibit defects in iron homeostasis, raising the question of the role of the Yfh1p processing defect in causing this abnormality. It now appears that defective maturation of Yfh1p is unlikely to cause mitochondrial iron accumulation in ssq1 mutants. The mutant Yfh1p(K49G, R50G) was efficiently imported and processed to generate only the intermediate form; the second MPP processing step was completely blocked and no mature form was generated, thereby mimicking the processing defect phenotype of the ssq1 mitochondria. However, the mutant Yfh1p intermediate with intact domain II was as efficient as the wild-type mature Yfh1p in complementing slow growth and iron accumulation phenotypes of the yfh1 mutant. The second processing step is therefore not critical for Yfh1p function. This is similar to what has been described for Rip1p processing. On import, Rip1p is processed in two steps and the second processing by MIP is not essential for import and assembly of functionally active Rip1p into the cytochrome bc1 complex (27).
Mapping the margins of cleaved domains of the Yfh1p precursor has allowed us to ascertain their functions. Domain I behaved like a typical matrix-targeting signal; it was sufficient for targeting and import of a non-mitochondrial passenger protein. Furthermore, domain I could be replaced by other matrix-targeting signals without affecting efficient import and functions of Yfh1p. In contrast to domain I, domain II by itself was not sufficient for import of a passenger protein. Interestingly, however, domain II was required for efficient Yfh1p import. It is unlikely that domain II serves as an intramolecular chaperone for Yfh1p because prior denaturation with urea cannot obviate the import defects of precursor proteins lacking domain II. How then does domain II participate in the import process? Domain II likely serves as a critical spacer separating domain I (or other mitochondrial targeting sequences) from the mature portion of Yfh1p. A typical matrix-targeting signal is rich in basic residues: domain I of Yfh1p, the Cox4p signal sequence and the Put2p signal sequence contain 5, 4 and 4 basic amino acids, respectively, and do not contain any acidic residues. On the other hand, the N-terminal portion of the mature Yfh1p [corresponding to residues 52–103 of the Yfh1p precursor (Fig. 1B)] is rich in acidic residues (16 Asp/Glu versus 1 Lys). When these acidic residues are placed immediately adjacent to a positively charged matrix-targeting signal as in Yfh1p1–22–mYfh1p, Cox4p1–22–mYfh1p and Put2p1–17–mYfh1p, non-specific ionic interactions may interfere with the targeting function of the signal sequence. The presence of the 31 amino acids of domain II between domain I and the mature protein could obviate these unwanted interactions, thereby allowing recognition of the targeting signal by the mitochondrial import receptors and/or subsequent passage through the import channels. The requirement for a spacer separating the matrix-targeting signal from the mature part of the precursor protein may not be unique to Yfh1p. Cleaved domains at the N-termini of mitochondrial precursor proteins vary in length and it remains to be seen whether some of the longer domains perform dual targeting and spacer functions.
What do our data imply regarding the relationship of processing of the human protein (frataxin) to its function? We have previously demonstrated that human frataxin (30 kDa) was efficiently imported into isolated mammalian and yeast (wild-type or ssq1 mutant) mitochondria. In all cases, the N-terminal signal sequence was removed, generating an 18 kDa form of frataxin. Unlike Yfh1p, no intermediate product migrating between the precursor and the 18 kDa form was detected during maturation of frataxin. Likewise, when frataxin was incubated with purified recombinant yeast or rat MPP, the products generated were identical in size to the 18 kDa form of frataxin observed during its maturation within mitochondria and no intermediate products were detected. The only other additional band detected as a result of MPP cleavage was the 4.2 kDa signal peptide. These results led us to conclude that frataxin was processed in one step by MPP (17). In contrast, import studies by Isaya and co-workers (13,18,28) have identified a 21 kDa protein as the major product generated during maturation of frataxin. On longer incubation, a minor amount of 18 kDa protein and other smaller degradation products were detected. Incubation of frataxin with recombinant MPP also resulted in the appearance of a major 21 kDa protein and a small amount of 18 kDa protein. These results led the authors to conclude that frataxin, like Yfh1p, is sequentially processed in two steps by MPP. The reason for these apparently discrepant data on frataxin processing is not clear. It could be that frataxin is indeed processed in one step: the 18 kDa protein in our gel system is the mature form and it runs slightly slower (21 kDa) in the gel system of others; the minor bands, including the 18 kDa band seen by others, may then represent non-specific degradation products. Alternatively, frataxin may be processed in two steps yet one of the steps may be undetected in our assay system, either because it was too efficient (leading to a single band of the mature protein) or too inefficient (yielding a single band of the intermediate). More work is needed to resolve this issue.
Disease-causing mutations in the C-terminal domain of frataxin have been identified with effects on expression, processing, stability and function of the protein (13,28,29). Immunoblot analysis of wild-type frataxin overexpressed in the yfh1 mutant revealed a major 21 kDa and a minor 18 kDa band. As in the case of in vitro import studies, these two bands were considered the intermediate and mature forms of frataxin, respectively (13). The levels of the 21 kDa bands were reduced by 4-fold for frataxin mutants, G130V or W173G. The 18 kDa bands varied from a low level (G130V) to a trace amount (W173G) and frataxin (G130V) was found to be more effective than frataxin (W173G) in complementing the yfh1 mutant. These results were considered to provide a correlation between impaired maturation and impaired function of frataxin (13). However, based on the crystal structure of frataxin (30) most disease-causing mutations (e.g. W173G) are likely to disrupt the hydrophobic core of the protein. This hydrophobic region has been postulated to play a critical role in the maintenance of a stable compact structure and perturbation of this domain is therefore likely to cause major changes in protein folding. These mutations are therefore not informative in evaluating the importance of processing for function. We have demonstrated that when the conversion of the intermediate to the mature form was completely blocked, the mutant Yfh1p(K49G, R50G) was able to efficiently complement the yfh1 phenotypes. A similar assay using frataxin that lacks the MPP cleavage site(s) should be useful for directly evaluating the relationship between its processing and functions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Constructs
pSP64T/Yfh1p1–53–Protein A.
The sequence encoding the first 53 amino acids of Yfh1p was amplified from pSP64T/Yfh1p (17) using the primers 5'-aaagcttaacatatgattaagcggtctctcgcaagt-3' (sense) and 5'-ccggtaccgaattcttctacaaatctcttctgaaaagtatttac-3' (antisense). The NdeI–Yfh1p1–53–KpnI fragment of the PCR product was subcloned into pET21b/Yfh1p–Protein A (14). The NdeI–XhoI fragment of the resulting plasmid pET21b/Yfh1p1–53–Protein A was then moved to pSP64T, thereby generating the plasmid pSP64T/Yfh1p1–53–Protein A.
pSP64T/Yfh1p1–22–Protein A.
The sequence encoding the first 22 amino acids of Yfh1p was amplified by PCR using the primers 5'-aaagcttaacatatgattaagcggtctctcgcaagt-3' (sense) and 5'-ccggtaccgaattctatcatatatcttctgcccattacag-3' (antisense) using pSP64T/Yfh1p as the template. The resulting NdeI–Yfh1p1–22–KpnI fragment was cloned in-frame with Protein A in pSP64T.
pSP64T/20Yfh1p.
The sequence encoding amino acids 21–174 of Yfh1p was generated by PCR using pSP64T/Yfh1p as the template and the primers 5'-gctctagacatatgatagcagcggcaggagg-3' (sense) and 5'-cgcggatccttactcgagttggcttttagaaatggccttc-3' (antisense). The resulting NdeI–20Yfh1p–XhoI fragment was cloned into the vector pSP64T.
pSP64T/Yfh1p21–53–Protein A.
The sequence encoding amino acids 21–53 of Yfh1p was amplified by PCR using the primers 5'-gctctagacatatgatagcagcggcaggagg-3' (sense) and 5'-ccggtaccgaattcttctacaaatctcttctgaaaagtatttac-3' (antisense). The resulting NdeI–Yfh1p21–53–KpnI fragment was cloned in frame with Protein A in pSP64T.
pSP64T/Yfh1p1–22–mYfh1p and pET21b/Yfh1p1–22–mYfh1p.
The sequence encoding the first 22 amino acids of Yfh1p was generated by PCR using the primers 5'-aaagcttaacatatgattaagcggtctctcgcaagt-3' (sense) and 5'-ccggtaccgaattctatcatatatcttctgcccattacag-3' (antisense). Likewise, the sequence encoding amino acids 52–174 of Yfh1p (mYfh1p) was generated by PCR using the primers 5'-gcgcatatggaattcgtagaatcctcgacagatggtcaag-3' (sense) and 5'-cgcggatccttactcgagttggcttttagaaatggccttc-3' (antisense). The NdeI–Yfh1p1–22–EcoRI and EcoRI–mYfh1p–XhoI fragment were then cloned into the NdeI–XhoI site of the pSP64T vector. The NdeI–XhoI fragment of the resulting plasmid pSP64T/Yfh1p1–22–mYfh1p was then moved to pET21b, thereby generating the plasmid pET21b/Yfh1p1–22–mYfh1p.
pSP64T/Put2p1–17–iYfh1p and pET21b/Put2p1–17–iYfh1p.
The sequence encoding amino acids 21–174 of Yfh1p (iYfh1p) was generated by PCR using the oligonucleotides 5'-gcgtctagaatgatagcagcggcaggaggag-3' (sense) and 5'-cgcggatccttactcgagttggcttttagaaatggccttc-3' (antisense). The sequence encoding amino acids 1–17 of Put2p was generated by PCR using the primers 5'-ccggatcccatatgctatcagcaaggtgcctcaaatc-3' (sense) and 5'-cgcggatcctctagagaaagatctcttgaagtatatag-3' (antisense). The NdeI–Put2p1–17–XbaI fragment and the XbaI–iYfh1p–XhoI fragment were ligated into the NdeI–XhoI sites of pSP64T. The NdeI–XhoI fragment of the resulting plasmid pSP64T/Put2p1–17–iYfh1p was then moved to pET21b, thereby generating the plasmid pET21b/Put2p1–17–iYfh1p.
pSP64T/Put2p1–17–mYfh1p and pET21b/Put2p1–17–mYfh1p.
The sequence encoding amino acids 52–174 of Yfh1p (mYfh1p) was generated by PCR using oligonucleotides 5'-gcgtctagagtagaatcctcgacagatggtcaag-3' (sense) and 5'-cgcggatccttactcgagttggcttttagaaatggccttc-3' (antisense). The resulting XbaI–mYfh1p–XhoI fragment was cloned into the XbaI–XhoI site of the pSP64T/Put21–17–iYfh1p. The NdeI–XhoI fragment of the resulting plasmid pSP64T/Put2p1–17–mYfh1p was then moved to pET21b, thereby generating the plasmid pET21b/Put2p1–17–mYfh1p.
pSP64T/Put2p1–32–mYfh1p.
The sequence encoding the first 32 amino acids of Put2p was amplified from the plasmid pSP64/Put2p (24) using the primers 5'-ccggatcccatatgctatcagcaaggtgcctcaaatc-3' (sense) and 5'-cgctctagaaggttcatttcttatgtgcttgggg-3' (antisense). The resulting NdeI–Put2p1–32–XbaI fragment was cloned into the NdeI–XbaI site of pSP64T/Put2p1–17–mYfh1p.
pSP64T/Put2p1–32–m*Yfh1p.
The sequence encoding amino acids 47–174 of Yfh1p (m*Yfh1p) was generated by PCR using oligonucleotides 5'-gggaattctctagatttcagaagagatttgtagaatcctcg-3' (sense) and 5'-cgcggatccttactcgagttggcttttagaaatggccttc-3' (antisense). The XbaI–m*Yfh1p–XhoI fragment was used to replace the XbaI–mYfh1p–XhoI fragment of pSP64T/Put2p1–32–mYfh1p.
pSP64T/Cox4p1–22–mYfh1p and pSP64T/Cox4p1–22–iYfh1p.
The NdeI–Cox4p1–22–XbaI fragment from pCox4p–DHFR (24) was cloned into the NdeI–XbaI sites of pSP64T/Put2p1–17–mYfh1p and pSP64T/Put2p1–17–iYfh1p, generating pSP64T/Cox4p1–22–mYfh1p and pSP64T/Cox4p1–22–iYfh1p, respectively.
pSP64T/Yfh1p(R18G, R19G).
Arginine residues at positions 18 and 19 of Yfh1p were changed to glycine residues using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer’s protocol. The plasmid pSP64T/Yfh1p was used as the template and the method required two oligonucleotides: 5'-gctctgtaatgggcggaggatatatgatagcagcggc-3' (sense) and 5'-gccgctgctatcatatatcctccgcccattacagagc-3' (antisense).
pSP64T/Yfh1p(K49G, R50G).
Lysine and arginine at positions 49 and 50, respectively, of Yfh1p were mutated to glycine residues as described above using oligonucleotides 5'-ctgtaaatacttttcaggggggatttgtagaatcctcgac-3' (sense) and 5'-gtcgaggattctacaaatcccccctgaaaagtatttacag-3' (antisense).
All constructs were verified by sequencing.
Bacterial expression, in vitro transcription, translation and import
Cultures of Escherichia coli BL21(DE3) cells carrying different plasmids (pET21b/Yfh1p1–22–mYfh1p, pET21b/Put2p1–17–iYfh1p or pET21b/Put2p1–17–mYfh1p) were induced by isopropyl-ß-D-thiogalactopyranoside in the presence of a mixture of [35S]Met and [35S]Cys (EXPRE35S35S; NEN Life Science Products) and the radiolabeled overexpressed proteins with a C-terminal His6 tag were purified by Ni-NTA agarose (Qiagen) in the presence of 8 M urea as described by Sepuri et al. (24). For in vitro transcription, pSP64T plasmids containing various constructs were linearized with BamHI and their transcription was carried out using Ribomax-SP6 kit (Promega). 35S-labeled precursor proteins were synthesized in reticulocyte lysate (Promega) using the supplier’s protocol.
In vitro import reactions (14,17,24,31) were performed using mitochondria isolated from Saccharomyces cerevisiae strains D273-10B (ATCC 24657), 61 and 191-33C (ssq1-4, previously called ssc2-1). Import reactions contained 4 mM ATP, 1 mM GTP and 5 mM NADH. For urea-denatured precursors, the final urea concentration in the import assay was 0.16 M. Following import, reaction mixtures were treated with trypsin (0.2 mg/ml) for 30 min at 0°C. To inactivate trypsin, samples were diluted with 20 mM HEPES–KOH pH 7.5, 0.6 M sorbitol, 0.1 mg/ml bovine serum albumin, 5 mg/ml soybean trypsin inhibitor, 100 U/ml Trasylol and 1 mM phenylmethylsulfonyl fluoride. Mitochondria were sedimented (15 000 g for 10 min at 4°C) and washed with 10% trichloroacetic acid. Samples were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and autoradiography.
Mapping the MPP cleavage sites
Escherichia coli BL21(DE3) cells carrying the plasmid pVG18 were used for co-expression of both 
and ß subunits of yeast MPP and the functional enzyme complex was purified and assayed as described by Gordon et al. (17) and Géli (32). Yfh1p–Protein A with a C-terminal His6 tag was expressed in bacteria, purified (14) and used for determining the MPP cleavage sites of Yfh1p. A large-scale import reaction was performed at 30°C for 20 min using D273-10B mitochondria (20 mg) and purified Yfh1p–Protein A (10 µg). Unimported chimeric precursor was removed by trypsin treatment as described above. Mitochondria were re-isolated and solubilized in 50 mM Tris–HCl pH 7.5 containing 0.15 M NaCl, 5 mM EDTA, 0.25% Triton X-100, 10% glycerol and protease inhibitors. In parallel, Yfh1p–Protein A (3 µg) was treated with purified MPP (5 µg) in the absence of mitochondria in 50 mM Tris–HCl pH 7.5 containing 0.15 M NaCl, 0.1% Triton X-100 and 10% glycerol. Following incubation at 30°C for 20 min, MPP was inactivated by the addition of EDTA (5 mM).
All samples were centrifuged at 128 000 g for 30 min at 4°C and each supernatant fraction was loaded onto a column containing 50 µl of rabbit IgG–Sepharose (5 mg/ml; Cappel). The column was sealed and rotated end over end at 4°C for 2 h. Following washing, the column was eluted with 0.3 M Tris base containing 3.9% SDS and 19% glycerol. The eluate was then treated with dithiothreitol (85 mM) at 55°C for 5 min. Proteins were separated by SDS–PAGE, blotted onto a PVDF membrane, stained with amido black and processed for N-terminal protein sequencing (33).
Genetic manipulations
Sporulation and tetrad dissection were performed according to standard methods (34).
Plasmids.
For interruption/deletion of YFH1, the plasmid yfh1 gamma was constructed by inserting 5' EagI–BamHI and 3' BamHI–XhoI genomic fragments of YFH1 into the vector pRS404. The covering plasmid, pRS318–YFH1, was constructed by subcloning the genomic HindIII fragment containing the YFH1 open reading frame (ORF) and promoter sequences into the same site of pRS318. The resulting vector contained LEU2 and CYH2 genes for selection and cycloheximide-dependent counterselection, respectively. The HindIII genomic fragment of the covering plasmid was also subcloned into pRS406 and NdeI and XhoI sites were introduced prior to and following the YFH1 ORF, respectively, using oligonucleotide-directed mutagenesis. To evaluate in vivo functions, various chimeric and mutant ORFs were introduced between NdeI and XhoI sites replacing YFH1 ORF in the plasmid pRS406/YFH1.
Generation of YFH1 shuffle strains.
A diploid strain resistant to cycloheximide, YPH501 (cyh2/cyh2), was generated as described by Li et al. (35). YPH501 was transformed with yfh1 gamma which had been linearized with BamHI and Trp+ transformants were selected. The diploid was then transformed with pRS318–YFH1 and Leu+ transformants were sporulated. A tetrad clone with Leu+Trp+ growth was identified and designated as the 6Ayfh1 shuffle strain.
Complementation.
To evaluate the in vivo complementation of various YFH1 constructs in pRS406, the corresponding plasmids were digested with StuI and integrated at the ura3-52 locus of the 6Ayfh1 shuffle strain. Transformants initially selected for Leu+Ura+ prototrophy were then transferred to rich media (YPAD) plates supplemented with cycloheximide (10 µg/ml). After 4 days, the covering pRS318–YFH1 plasmid was ejected as shown by the loss of leucine prototrophy and these strains were analyzed for high-affinity iron uptake (14,35,36). To evaluate mitochondrial function, these cells were also streaked on solid YE media (1% yeast extract, 2% peptone, 2% agar and 3% ethanol). The YFH1 mutant alleles expressed in the 6Ayfh1 shuffle strain after ejection of the covering plasmid were grown in raffinose-based medium (1% yeast extract, 2% peptone, 0.01% adenine, 2% raffinose and 0.1% glucose) and mitochondria were isolated.
Antibodies
Yfh1p precursor with a C-terminal His6 tag was expressed in bacteria and purified using Ni-NTA chromatography (14). Anti-Yfh1p antibodies were raised in rabbits against the purified precursor protein. Other antibodies used in this study have been described elsewhere (31,37). For western blots, mitochondrial proteins (100 µg) were separated by SDS–PAGE and blotted to nitrocellulose. The primary antibodies were rabbit polyclonal antibodies and the signal was developed using anti-rabbit IgG conjugated to peroxidase and the ECL kit (Amersham).
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ACKNOWLEDGEMENTS |
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We thank Vincent Géli for the plasmid pVG18. Protein sequence data were obtained from the Rockefeller University Protein/DNA Technology Center. This work was supported by grants from the NIH (GM57067) and the American Heart Association (9951300U) to D.P. A.D. is supported by the NIH grant DK53953. D.M.G. is supported by the NRSA fellowship NS11166.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 215 573 7305; Fax: +1 215 573 5851; Email: pain@mail.med.upenn.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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