From the
Adolf-Butenandt-Institut für Physiologische Chemie,
Ludwig-Maximilians-Universität München, Butenandtstrasse 5, 81377
München, Germany and the
Max-Delbrück-Centrum für Molekulare
Medizin, Robert-Rössle-Strasse 10, 13092 Berlin/Buch, Germany
Received for publication, November 5, 2002 , and in revised form, March 31, 2003.
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ABSTRACT |
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INTRODUCTION |
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Dynamins are a family of large GTPases involved in membrane fission during endocytosis (reviewed in Ref. 14). However, the physiological roles of the mitochondrial dynamin-like proteins Mgm1 and OPA1 are not understood very well. Several hypotheses have been put forward that are mainly based on the homology to dynamins, on the mitochondrial localization, and the fragmentation of mitochondria in strains in which Mgm1 is mutated or deleted (1012). According to one hypothesis, Mgm1 modulates the morphology of the inner membrane and therefore is thought to be involved in cristae formation and/or inner membrane fission events (2). Down-regulation of OPA1 in HeLa cells by small interfering RNA was reported to alter cristae morphology (15). On the other hand, two recent reports indicate an important role for Mgm1 in the fusion of mitochondria (3, 4). Contradicting views exist about the exact submitochondrial location of Mgm1. It is still a matter of debate whether Mgm1 is located in the outer membrane (12) or in the intermembrane space of mitochondria (24) and whether its Schizosaccharomyces pombe ortholog, Msp1, resides in the matrix (16). Mgm1 and its orthologs from all species investigated so far exist in at least two isoforms (2, 12, 1719). Thus, it is crucial to clarify the identity of these isoforms and how they are generated. Furthermore, it is important to unravel the function of each of these isoforms. Are they both necessary for wild type mitochondrial morphology and maintenance of mitochondrial DNA, or do they serve independent functions? Here we present data that provide new insights into the biogenesis of Mgm1 and the role of its two isoforms for wild type-like mitochondrial morphology and maintenance of mtDNA1 in yeast.
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EXPERIMENTAL PROCEDURES |
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AntibodiesAntibodies against Mgm1 were raised in rabbits using as antigens the C-terminal peptide H2N-CKKSYKGVSKNL-COOH and the internal peptide H2N-CSHQFEKAYFKENKK-COOH, both containing an additional cysteine for coupling to an affinity resin. Peptide synthesis, coupling of the peptide to keyhole limpet hemocyanin, and immunization of the rabbits were carried out by Pineda Antikörperservice (Berlin, Germany). For affinity purification, peptides were coupled to SulfoLink® Coupling Gel (Pierce) according to the manufacturer's instructions. The antibody against the C-terminal epitope was used for Western analysis unless indicated differently.
Determination of Growth on Nonfermentable Carbon SourceIn order to compare growth on fermentable versus nonfermentable carbon sources, drop dilution assays were performed. After tetrad dissection, cells were grown to exponential phase for 16 h on liquid-selective glucose medium at 30 °C, adjusted to a concentration of 0.7 A578 nm/ml, and subjected to consecutive 10-fold dilution steps. 5-µl aliquots of each dilution were spotted on YPD and YPG plates in duplicate, and the plates were incubated for two (YPD) or three (YPG) days at 30 °C.
Import of Preproteins into MitochondriaRadiolabeled precursor proteins were synthesized using a coupled reticulocyte lysate transcription-translation system (Promega) in the presence of [35S]methionine. Mitochondria were isolated as described (23). Import reactions were carried out in import buffer at 25 °C (600 mM sorbitol, 50 mM HEPES, 80 mM KCl, 10 mM MgAc2, 2.5 mM EDTA, 2 mM KH2PO4, 5 mM NADH, 2.5 mM ATP, 2.5 mM malate, 2.5 mM succinate, 0.1% bovine serum albumin, pH 7.2). 50 µg of mitochondria and 1% (v/v) reticulocyte lysate with the radiolabeled precursor were used per import reaction. Membrane potential was dissipated by adding carbonyl cyanide 3-chlorophenylhydrazone to a final concentration of 50 µM. After import, samples were diluted, treated with hypoosmotic buffer (20 mM HEPES/KOH, pH 7.4) to selectively rupture the outer membrane, and treated with proteinase K as indicated.
Carbonate ExtractionTo extract peripherally bound membrane proteins, mitochondria were diluted to a final concentration of 1 mg/ml in 20 mM HEPES/KOH, pH 7.4. After the addition of an equal volume of freshly prepared 0.2 M sodium carbonate solution, samples were incubated for 30 min at 4 °C. The membrane and soluble fractions were separated by centrifugation at 45,000 rpm in a TLA45 rotor for 30 min at 4 °C. Equal fractions of membrane-associated and soluble proteins were analyzed by SDS-PAGE and immunoblotting.
Yeast Total Cell ExtractsYeast total cell extracts were
prepared by alkaline lysis. The pellet of 2 ml of yeast culture
(A578 = 1) was resuspended in 250 µl of 50
mM Tris/HCl, pH 8. Then 50 µl of lysis buffer (1.85 M
NaOH, 7.4% (v/v) -mercaptoethanol and 20 mM
phenylmethylsulfonyl fluoride) were added. After incubation for 10 min at 4
°C, samples were precipitated with 220 µl of 72% (w/v) trichloroacetic
acid, washed once with acetone, and analyzed by SDS-PAGE and Western
blotting.
Fluorescence MicroscopyHeterozygous diploid strains were cotransformed with plasmid pVT100U-mtGFP expressing mitochondria targeted green fluorescent protein (24). After tetrad dissection, cells were grown for 16 h to exponential phase in liquid-selective glucose medium at 30 °C and analyzed by standard fluorescence microscopy (25). Classification and quantification of the morphology phenotypes were performed without knowledge of strain identity at the time of analysis. To test for the presence of mitochondrial DNA, cells grown under the same conditions were stained with 1 µg/ml DAPI for 1 h and washed once with phosphate-buffered saline. Only cells showing no trace of staining outside the nucleus were classified as lacking mtDNA (rhoO). For quantification of the phenotype, at least 150 cells were analyzed in three independent experiments, and the average and S.D. were calculated.
Determination of N Termini of Mgm1 Isoforms10 mg of mitochondria were lysed in 10 mM Tris/HCl, pH 7.6, 0.5% (w/v) Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride for 15 min. After a clarifying spin, the supernatant was subjected to immunoprecipitation for 3 h at 4 °C using Protein A-Sepharose beads (Amersham Biosciences) preloaded with antibodies against the C terminus of Mgm1. Samples were eluted from the beads with SDS-containing buffer, separated by SDS-PAGE, and blotted onto a polyvinylidene difluoride membrane. Mgm1 bands were cut and subjected to N-terminal sequencing by Edman degradation (TOPLAB GmbH, Germany).
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RESULTS |
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In a second approach, we studied the submitochondrial distribution of endogenous Mgm1. Isolated yeast mitochondria were treated with proteinase K and subjected to SDS-electrophoresis and immunoblotting using antibodies directed against a C-terminal (Fig. 1C) and an internal epitope of Mgm1 (Fig. 1D). Endogenous Mgm1 was protected from digestion by proteinase K in mitochondria in both cases. After selective opening of the outer membrane, both isoforms of Mgm1 were digested by the protease. Swelling efficiency and the intactness of the inner membrane after swelling were controlled by decorating the same Western blots with antibodies against Oxa1 and Aac2; fragments characteristic for mitochondria with opened outer membrane and intact inner membrane were observed (26, 27). Taken together, the C terminus and the internal epitope (residues 484497) of both Mgm1 isoforms are located in the intermembrane space. This is consistent with the localization proposed by Wong et al. (2) and by two recent reports (3, 4). Also, the human ortholog OPA1 was recently localized to this subcompartment (17).
In summary, both isoforms of Mgm1, the large isoform (l-Mgm1; 97 kDa) and the short isoform (s-Mgm1; 84 kDa) are located in the intermembrane space.
Determination of the N Termini of Both Mgm1 IsoformsIn order to further analyze the structure of the two isoforms, their N termini were determined. To this end, mitochondria were solubilized, and Mgm1 was immunoprecipitated using an antibody raised against the C terminus. After SDS-PAGE and blotting onto a polyvinylidene difluoride membrane, the bands corresponding to the two isoforms were cut out from the membrane, and the respective N termini were determined by Edman degradation (Fig. 2A). In the case of l-Mgm1, the N-terminal sequence was ISHFPKII, corresponding to amino acid residues 8188 of Mgm1. Since there is an MPP consensus site (28) after residue Ser80, we conclude that l-Mgm1 is generated by MPP cleavage immediately after the mitochondrial targeting sequence of Mgm1. In the case of s-Mgm1, the two peptides ATLIAATS and LIAATS were found in similar amounts. These peptides correspond to amino acid residues 161168 and 163168, respectively. We suggest that the initial cleavage takes place after Thr160 and that the further removal of two residues is caused by other peptidases in the intermembrane space or during the preparation of cell extracts. Still, the possibility is not excluded that the initial cleavage of Mgm1 can occur after Thr160 as well as after Thr162.
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Membrane Association of Mgm1Mgm1 contains a predicted transmembrane segment from residue 94 to 113, which, as shown above, is only present in l-Mgm1 and not in s-Mgm1. Therefore, the two isoforms should differ in their membrane association. In order to address this, mitochondria were swollen to rupture the outer membrane and then extracted with either low salt, high salt, or sodium carbonate, and membranes were separated by ultracentrifugation. Neither of the two isoforms of Mgm1 was released from mitochondria by swelling alone, indicating that they are not soluble proteins in the intermembrane space (Fig. 2B). With high salt, about 50% of s-Mgm1 was released from the membranes, whereas l-Mgm1 was resistant to salt extraction (Fig. 2B). Treatment with sodium carbonate led to the removal of significant fractions of both isoforms from the membrane (Fig. 2B). However, l-Mgm1 was more resistant, probably because it contains the predicted N-terminal membrane-spanning segment. The incomplete resistance of l-Mgm1 to carbonate extraction can be attributed to the presence of only few amino acid residues on the N-terminal side of the predicted transmembrane segment, leading to a weaker embedding in the membrane. A similar behavior upon alkaline treatment of mitochondria was observed for D-lactate dehydrogenase (Fig. 2B), a protein that is anchored to the inner membrane by a single N-terminal transmembrane segment and faces the intermembrane space (29). In contrast, the ADP/ATP carrier (Aac2), an integral membrane protein with six membrane-spanning segments (27), was completely resistant to extraction by carbonate (Fig. 2B). In order to investigate whether l-Mgm1 and s-Mgm1 differ in their association with either the outer or the inner membrane, we generated mitochondrial membrane vesicles by sonication and fractionated them on a sucrose gradient. Both isoforms of Mgm1 were found to be associated with vesicles derived from both membranes (data not shown). This may be due to the release of Mgm1 from membranes during sonication and subsequent nonspecific binding to vesicles. Such an association with membranes would not be unusual for a dynamin-like protein, since dynamins are known to bind to and spontaneously assemble on lipid vesicles (30). Unfortunately, this behavior makes it highly unreliable to conclude to which membrane Mgm1 is attached.
The Mitochondrial Protease Pcp1 Is Involved in the Generation of
s-Mgm1In order to investigate how the isoforms of Mgm1 are
generated, we prepared total cell extracts of different deletion mutants that
were deficient in one of the known or putative mitochondrial proteases. In all
strains, similar steady state levels of both isoforms of Mgm1 as in wild type
were observed, with the exception of the pcp1 strain
(Fig. 3). In this strain, the
band corresponding to s-Mgm1 was absent, whereas the steady state level of
l-Mgm1 was increased. Two additional minor bands were observed in the
pcp1 strain, but they were distinct from s-Mgm1 in size. They
are probably the result of nonspecific degradation during the preparation of
cell extracts. Thus, the presence of Pcp1 appears to be essential for the
generation of s-Mgm1.
Pcp1 was shown to be imported into mitochondria in vitro
(31). Furthermore, Pcp1 was
identified in a recent screen for mutants with aberrant mitochondrial
morphology and predicted to be located in the inner membrane of mitochondria
(32). In that study, the
pcp1 strain showed a phenotype strikingly similar to that of
the
mgm1 strain; both strains have fragmented mitochondria and
cannot grow on nonfermentable carbon sources
(10,
11). The
mgm1
strain is rho0, since it has lost mtDNA
(11). We checked the presence
of mtDNA in the homozygous diploid
pcp1/
pcp1
and
mgm1/
mgm1 strains by DAPI staining and
fluorescence microscopy. Indeed, 94.0 ± 3.1% of
pcp1/
pcp1 and 95.4 ± 1.5% of
mgm1/
mgm1 cells (n > 150) were
classified as rho0 by DAPI staining and fluorescence microscopy
(wild type W303: 1.0 ± 0.5%). Since classification was done in a
conservative fashion, presumably all cells were lacking mtDNA. Pcp1 contains a
domain with sequence similarity to Rhomboid, a serine protease in
Drosophila melanogaster
(33), and was reported to be
required for proteolytic processing of cytochrome c peroxidase in the
intermembrane space (34).
Therefore, the phenotype of
pcp1 is likely to be a direct
consequence of deficient and/or improper proteolytic processing of l-Mgm1 to
s-Mgm1. Still, Pcp1 may be involved in the processing of further proteins or
may even exert other cellular functions that might be equally important for
the maintenance of mitochondrial morphology and of mtDNA.
Complementation Analysis of s-Mgm1 in the pcp1
StrainIs the phenotype observed upon deletion of PCP1 a
direct consequence of the failure to generate s-Mgm1 or a different effect
independent of Mgm1? We checked whether expression of the short isoform of
Mgm1 under its endogenous promoter can complement the phenotype of the
pcp1 strain. To achieve import into the intermembrane space,
the targeting and sorting signals of cytochrome b2
(35) were fused to the
determined N terminus of s-Mgm1, thereby generating the precursor
pb2167s-Mgm1. Upon import of cytochrome b2 into
mitochondria, the N terminus is cleaved by MPP and the mitochondrial inner
membrane protease, yielding a mature protein in the intermembrane space
lacking the first 80 amino acid residues
(35). Thus, upon import of
pb2167s-Mgm1, the short form of Mgm1 containing an N-terminal
extension of residues 81167 of cytochrome b2 is
generated (Fig. 4A).
This fusion protein has roughly the same size as l-Mgm1. We refer to it as
s-Mgm1*, since it contains only residues 161902 of Mgm1. We transformed
a heterozygous diploid PCP1/
pcp1 strain with a
plasmid coding for s-Mgm1* under the endogenous Mgm1 promoter, since the
homozygous diploid
pcp1/
pcp1 strain has
irreversibly lost mtDNA. Tetrads were obtained after sporulation, and haploid
spores were tested for their phenotype. First, we checked for the presence of
s-Mgm1* in total cell extracts. A protein band of the expected size was
detected with cells containing the plasmid coding for s-Mgm1*
(Fig. 4B). It has to
be noted that the size of s-Mgm1* is identical to the size of the endogenous
l-Mgm1, but an increase in signal intensity upon expression of s-Mgm1* is
evident. Expression levels of s-Mgm1* varied to a certain extent between
different strains obtained from individual spores, but they were comparable
with those of endogenous s-Mgm1 in the wild type strain.
(Fig. 4B).
Next we determined whether expression of s-Mgm1* can rescue the
respiration-deficient phenotype of the pcp1 strain. Cell
growth was analyzed by drop dilution assays in parallel on YPD (fermentable
carbon source) and YPG plates (nonfermentable carbon source).
pcp1 strains carrying the plasmid coding for s-Mgm1* grew on
YPG in contrast to the
pcp1 strain lacking it
(Fig. 4C). The extent
of growth on YPG varied between the three strains shown and was highest in
strain
pcp1+s-Mgm1*-1, having the highest expression level of
s-Mgm1*. Still, growth on YPG of the latter cells was slower than that of
PCP1 wild type cells (Fig.
4C). This indicates that s-Mgm1* can partially complement
the respiration-deficient phenotype of the
pcp1 strain. Since
overexpression of s-Mgm1* in a wild type background resulted in a
dominant-negative phenotype (data not shown), we investigated whether also
wild type-like levels of s-Mgm1* had any dominant negative effect. This was
not the case, since growth of wild type PCP1 cells on YPD or YPG was
not altered upon expression of s-Mgm1*
(Fig. 4C). In
addition, mitochondrial morphology and the maintenance of mtDNA were not
affected in these strains (Fig. 5,
A and B, and
Table I).
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We asked whether loss of respiration competence was correlated with loss of mitochondrial DNA. Indeed, also maintenance of mtDNA was dependent on the expression of s-Mgm1* as judged by DAPI staining and fluorescence microscopy (Table I). Loss of mtDNA was suppressed only partially by s-Mgm1* compared with wild type controls. In addition, loss of mtDNA was less pronounced in cells that showed faster growth in the drop dilution assays on YPG. We conclude that the short form of Mgm1 used in our experiments, s-Mgm1*, is functional and can partially suppress the loss of mtDNA in a strain lacking the rhomboid-type protease Pcp1. A clear correlation between respiration competence and maintenance of mtDNA was observed (Table I). However, mechanisms other than loss of mtDNA may contribute to explaining how respiration competence was lost in some strains containing low but significant amounts of mtDNA.
In order to test whether expression of s-Mgm1* can alleviate the defect in
mitochondrial morphology of the pcp1 strain, mitochondria were
visualized by fluorescence microscopy using mitochondria-targeted green
fluorescent protein, and the percentage of cells showing a wild type-like
mitochondrial tubular network was scored. Cells showing fragmented or
aggregated mitochondria were classified as non-wild type-like. The percentage
of
pcp1 cells with wild type-like mitochondrial morphology was
increased significantly only when s-Mgm1* was expressed
(Fig. 5, A and
B, and Table
I). Nevertheless, complementation of the morphology defect was
only partial and was not observed for the strain
pcp1+s-Mgm1*-1, although this strain complemented the
respiration defect and the loss of mtDNA to the highest extent among all three
strains of this kind (Table I).
There was variation between the different strains we investigated, but in
general expression of s-Mgm1* led to a restoration of the mitochondrial
morphology defect compared with
pcp1 cells lacking s-Mgm1*.
The percentage of wild type-like cells was never higher than 13.7 ±
3.1% in four
pcp1 strains lacking s-Mgm1*, whereas for strains
expressing s-Mgm1* we observed up to 30.0 ± 3.2% wild type-like cells
(
pcp1+s-Mgm1*-3).
Taken together, s-Mgm1* is functional, and expression of s-Mgm1* can
partially suppress the mitochondrial morphology defect as well as the loss of
respiration competence and the loss of mitochondrial DNA of the
pcp1 strain.
Functional Analysis of Mgm1 IsoformsIs only one or are both
isoforms of Mgm1 required for the integrity of mitochondria? We expressed the
two isoforms either separately or both together in the mgm1
strain. For expression of s-Mgm1*, the same plasmid as described above was
used; for expression of l-Mgm1, a variant was used in which the cleavage
region was deleted to prevent formation of s-Mgm1. The whole region was
deleted, since a defined cleavage signal for Pcp1 could not be recognized.
This version of Mgm1 is referred to as l-Mgm1*
(Fig. 4A). A
heterozygous diploid MGM1/
mgm1 strain was transformed
either with a plasmid coding for s-Mgm1*, with a plasmid coding for l-Mgm1*,
or with both of these plasmids. As controls, either no plasmid or a plasmid
coding for the full-length Mgm1 was used. In all cases, expression was under
control of the endogenous Mgm1 promoter. Tetrads were obtained upon
sporulation, and the phenotype of haploid spores was analyzed. First, we
checked for the expression of Mgm1, s-Mgm1*, and l-Mgm1* in total cell
extracts. Expression of all constructs was checked by Western blotting
(Fig. 4D). Due to
deletion of the cleavage region, l-Mgm1* is smaller than endogenous l-Mgm1,
and its size is identical to that of the putative degradation product of
s-Mgm1* (Fig. 4D).
Expression levels of all constructs were similar to those of endogenous s-Mgm1
and l-Mgm1 in wild type cells. Furthermore, the expressed proteins were
correctly located in the intermembrane space of mitochondria as determined by
immunoelectron microscopy, cell fractionation, and proteinase K protection
experiments (data not shown).
Can the expression of Mgm1, l-Mgm1*, s-Mgm1*, or l-Mgm1* and s-Mgm1*
together rescue the respiration-deficient phenotype of the
mgm1 strain? In the
mgm1 background, no growth
on YPG was observed when only one of the two isoforms of Mgm1 was present
(Fig. 4E). This lack
of growth correlates well with the pronounced loss of mitochondrial DNA in
mgm1 strains expressing either s-Mgm1* or l-Mgm1* alone
(Table I). Likewise, neither
s-Mgm1* nor l-Mgm1* rescue the defective mitochondrial morphology of the
mgm1 strain when expressed alone
(Fig. 5, A and
B, Table
I). In contrast, complementation of the respiration-deficient
phenotype did occur when l-Mgm1* and s-Mgm1* were coexpressed
(Fig. 4E).
Complementation as measured by growth on YPG was, however, only partial with
the strain
mgm1+l&s-Mgm1*-3 and not observed with the
strains
mgm1+l&s-Mgm1*-1 and
mgm1+l&s-Mgm1*-2. Nevertheless, in all three strains, loss
of mtDNA was significantly suppressed
(Table I). Finally, we checked
whether coexpression of l-Mgm1* and s-Mgm1* also complements the mitochondrial
morphology defect in these strains. Interestingly, the strains
mgm1+l&s-Mgm1*-1 and
mgm1+l&s-Mgm1*-2,
which did not grow on YPG, showed significant suppression of the mitochondrial
morphology defect (Fig. 5, A and
B, and Table
I).
Differences in the expression levels of s-Mgm1* and l-Mgm1* or the ratio of
both isoforms may lead to different degrees of complementation. Indeed, the
expression levels of the isoforms in the pcp1 as well as in
the
mgm1 background varied among strains obtained from
different spores (Fig. 4, B and
D). Interestingly, in both backgrounds, restoration of
the mitochondrial morphology defect was inversely correlated to the
suppression of respiration competence and loss of mtDNA. It seems that
expression levels are very critical and affect loss of mtDNA and mitochondrial
morphology differently. Nonetheless, both variants of the Mgm1 isoforms are
functional, and both isoforms are necessary to partially complement defects in
mitochondrial morphology as well as loss of respiration competence and of
mtDNA.
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DISCUSSION |
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We have addressed here the identity and role of the two isoforms of Mgm1. The two isoforms have been described to be present in roughly equal yet variable amounts under steady-state conditions in yeast (2, 12). In mice, the ratio between the isoforms was found to vary among different tissues (18). The two isoforms of yeast Mgm1 differ in the presence of an N-terminal segment of 80 amino acid residues. This segment contains a hydrophobic stretch predicted to be a transmembrane domain; it is present in all orthologs from other organisms. In addition, we have localized Mgm1 by immunoelectron microscopy to the cristae membrane as well as to the region where inner and outer membrane are closely apposed,2 confirming that Mgm1 is associated at least to some extent with the inner membrane. On the basis of available data, we suggest a topogenesis pathway in which the presequence of the precursor of Mgm1 becomes exposed to the matrix and the N-terminal hydrophobic segment becomes anchored in the inner membrane by a translocation-arrest mechanism. Cleavage by MPP then generates the large isoform of Mgm1, l-Mgm1. Subsequently, part of l-Mgm1 undergoes further proteolytic cleavage. The product, s-Mgm1, becomes released into the intermembrane space and subsequently attached to the outer or the inner membrane. Direct processing of the arrested precursor of Mgm1 to s-Mgm1, however, cannot be excluded.
We report here that Pcp1 is involved in the processing of Mgm1. Deletion mutants of PCP1 contain fragmented and short tubular mitochondria. The Pcp1 protein was predicted to reside in the inner membrane of mitochondria (32). One feature of Pcp1 is a domain with sequence similarity to Rhomboid, a serine protease in D. melanogaster (33). Pcp1 is involved in the processing of cytochrome c peroxidase (34). Cleavage of cytochrome c peroxidase by Pcp1 occurs directly after its hydrophobic sorting sequence, which has been suggested to serve as a translocation-arrest signal that becomes inserted into the inner membrane. Thereby, cytochrome c peroxidase becomes localized to the intermembrane space (36, 37). During maturation, cytochrome c peroxidase is initially cleaved by Yta10/Yta12 (34), a mitochondrial AAA-protease anchored in the inner membrane of which the active site resides in the matrix (38). As shown here, the processing of Mgm1 is not dependent on Yta10/Yta12. Therefore, processing by Yta10/Yta12 appears not to be a general prerequisite for proteolytic processing by Pcp1.
The phenotypes of the mgm1 and the
pcp1
strain are indistinguishable from each other. Both strains show fragmented and
short tubular mitochondria
(32) and lack mtDNA
(10,
11)
(Table I). Cells lacking the
Pcp1 protease still contain l-Mgm1 but lack s-Mgm1. Therefore, it is
conceivable that the deficiencies of the
pcp1 cells are caused
solely by the absence of the s-Mgm1 isoform. A functional variant of l-Mgm1
did not complement the
mgm1 phenotype. Thus, l-Mgm1 cannot
replace s-Mgm1 in its function. On the other hand, s-Mgm1 alone is not able to
take over the role of l-Mgm1, since it was not possible to complement the
mgm1 phenotype with a functional variant of s-Mgm1. We
conclude that both isoforms of Mgm1 are necessary for normal mitochondrial
morphology, respiration competence, and maintenance of mtDNA.
The Pcp1 protease may be involved in the biogenesis of further proteins
that are also important for respiration competence, maintenance of mtDNA, and
mitochondrial morphology. We consider this unlikely, since the
mgm1 phenotype was complemented by coexpression of the two
Mgm1 isoforms to a similar extent as the
pcp1 phenotype by
expression of the short isoform. In
mgm1 cells coexpressing
the two Mgm1 isoforms, the Pcp1 protease should process all other substrates
normally; in this case, one would expect that complementation is complete or
at least more pronounced compared with cells lacking Pcp1 and expressing the
short isoform. All of the phenotypic effects observed can currently be
explained without claiming a requirement of Pcp1 for processing of other
substrates relevant for the maintenance of mtDNA and mitochondrial morphology.
In agreement with this, the deletion of cytochrome c peroxidase, the
only other substrate for Pcp1 described so far
(34), did not show any defects
in mitochondrial morphology or respiration competence
(32). In conclusion, we
propose that the phenotype of the
pcp1 strain is due to a
failure of correct processing of Mgm1, although a possible role of other
substrates of Pcp1 cannot entirely be ruled out.
Pcp1 has sequence similarity to Rhomboid-type serine proteases, which are a large family of proteases found in eukaryotes, prokaryotes, and archaea (33, 39). Rhomboid from Drosophila triggers epidermal growth factor receptor signaling by proteolytic cleavage of precursors of epidermal growth factor receptor ligands, which are anchored to the plasma membrane by a single transmembrane segment (33, 39). After release, the ligands become soluble, are secreted, bind to the epidermal growth factor receptor of neighboring cells, and thereby activate this signaling pathway. Pcp1 may also be a protease exerting a signaling function although not between cells but instead between mitochondria or between mitochondrial membranes. The signaling by the Rhomboid-type protease Pcp1 could be through the short isoform of Mgm1. So far, two proteins involved in the fusion of mitochondria, Fzo1p and Ugo1p, have been proposed to interact with Mgm1 (3, 4). These proteins are potential candidates as receptors for s-Mgm1. On the other hand, s-Mgm1 might actively take part in a process such as fusion of mitochondria or cristae formation, in contrast to solely regulating it. In either case, proteolytic cleavage by Pcp1 seems to be an important upstream event.
The balanced formation or regulated interconversion of the two isoforms of Mgm1 appear crucial for mitochondrial morphology, respiration competence, and maintenance of mtDNA. What might be the molecular basis for the fact that both isoforms are required in parallel? One possibility is that they interact with each other and that altering the relative levels interferes with the formation and/or activity of such a hetero-oligomeric complex. Mgm1 was reported to be involved in the fusion of mitochondria (1, 3, 4). A hetero-oligomeric complex between s-Mgm1 and l-Mgm1 might play a role in the fusion of mitochondria and/or the coordinated fusion of the inner and the outer membrane. Another possibility is that both isoforms have separate functions equally important for mitochondrial morphology and maintenance of mtDNA.
In conclusion, we have presented data that provide new insights into the biogenesis of the dynamin-like protein Mgm1 and the role of its two isoforms for wild type-like mitochondrial morphology and maintenance of mtDNA in S. cerevisiae. The identification of Pcp1 as a membrane-integrated processing protease for Mgm1 has revealed a new unexpected aspect of limited proteolysis in mitochondria.
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FOOTNOTES |
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To whom correspondence should be addressed. Tel.: 49-89-2180-77100; Fax:
49-89-2180-77093; E-mail:
Andreas.Reichert{at}bio.med.uni-muenchen.de.
1 The abbreviations used are: mtDNA, mitochondrial DNA; DAPI,
4',6-diamidino-2-phenylindole; MPP, mitochondrial processing peptidase;
s-Mgm1, short isoform of Mgm1; l-Mgm1, large isoform of Mgm1.
2 M. Herlan, F. Vogel, C. Bornhövd, W. Neupert, and A. S. Reichert,
unpublished results.
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ACKNOWLEDGMENTS |
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REFERENCES |
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