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Report |
Address correspondence to Andreas S. Reichert, Adolf-Butenandt-Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, Butenandtstr. 5, 81377 München, Germany. Tel.: 49-89-2180-77100. Fax: 49-89-2180-77093. email: Andreas.Reichert{at}bio.med.uni-muenchen.de
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Abstract |
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Key Words: mitochondrial fusion; protein import; mitochondrial diseases; rhomboid protease; dynamin-like protein
Abbreviations used in this paper: Ccp1, cytochrome c peroxidase; DHFR, dihydrofolate reductase; l-Mgm1; large isoform of Mgm1; mtDNA, mitochondrial DNA; s-Mgm1, short isoform of Mgm1.
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Introduction |
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Results and discussion |
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A functional import motor is crucial for formation of s-Mgm1 and mitochondrial morphology
The first transmembrane segment of Mgm1 may act as a stop transfer signal during import of Mgm1 into mitochondria. Cleavage of the targeting signal by the mitochondrial processing peptidase leads to l-Mgm1, which is anchored to the inner membrane via this segment (Herlan et al., 2003). To check whether the balance between both isoforms is established already at the level of import of the precursor protein, we investigated whether down-regulation of essential components of the import motor of the inner mitochondrial membrane shifts the ratio of the two Mgm1 isoforms. Tim44 and Tim14 are such components. Together with Ssc1, the mitochondrial Hsp70 in yeast, and its nucleotide exchange factor Mge1, they mediate the ATP-driven import of preproteins into the mitochondrial matrix and the inner membrane (Neupert and Brunner, 2002; Mokranjac et al., 2003). Indeed, down-regulation of Tim44 and of Tim14 resulted in a substantial reduction in the formation of s-Mgm1 (Fig. 2 A), which is paralleled by increased fragmentation of mitochondria (Fig. 2, B and E). To rule out that reduced levels of the rhomboid protease Pcp1 caused decreased proteolysis of Mgm1, we determined the processing efficiency of Ccp1, the only other known substrate of Pcp1 (Esser et al., 2002). Upon down-regulation of Pcp1, accumulation of the intermediate form of Ccp1 and decreased levels of s-Mgm1 occur simultaneously showing that processing of Ccp1 and of Mgm1 are affected to a similar extent (Fig. 2 A; Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200403022/DC1). Down-regulation of Tim14 or Tim44 resulted in reduced Ccp1 levels at late time points but as no intermediate was observed Ccp1 processing was not impaired (Fig. 2 A). In this case, Pcp1 is not limiting for the formation of s-Mgm1. We conclude that Tim14 and Tim44 are necessary for the formation of s-Mgm1. Tim17 is an essential subunit of the TIM23 preprotein conducting channel of the inner membrane (Neupert and Brunner, 2002). Down-regulation of Tim17 only had a mild effect on the formation of s-Mgm1 and similarly affected the formation of l-Mgm1 (Fig. 2 A). Thus, the import channel is required for the formation of either isoform of Mgm1. Reduced import of both Mgm1 isoforms and potentially of other components essential for wild-type mitochondrial morphology are most likely the reason for the effects on mitochondrial morphology upon down-regulation of Tim17 (Fig. 2, B and E). Tim22 is essential for import of proteins with internal signal sequences such as the ADP/ATP carrier (Sirrenberg et al., 1996). Mgm1 is synthesized as a precursor with an NH2-terminal targeting sequence and therefore unlikely to be a substrate for Tim22. Indeed, down-regulation of Tim22 neither affected Mgm1 biogenesis nor mitochondrial morphology. No component essential for wild-type mitochondrial morphology seems to require the Tim22 import pathway into the inner membrane. Moreover, the reduction of s-Mgm1 levels is not a general consequence of down-regulating an essential mitochondrial protein.
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Formation of s-Mgm1 but not of l-Mgm1 is ATP dependent
To further investigate topogenesis of Mgm1 isoforms in vitro, radiolabeled variants of Mgm11-228dihydrofolate reductase (DHFR) precursors were imported into isolated yeast mitochondria and subsequently treated with trypsin. After import, bands corresponding to l- and s-Mgm11-228DHFR were observed (Fig. 3 A). Consistent with the results obtained in vivo (Fig. 1 B) formation of s-Mgm11-228DHFR was increased with variants in which the first hydrophobic segment was more hydrophilic (Fig. 3 A, G100D, G100K). No formation of s-Mgm11-228DHFR was observed when it was more hydrophobic (Fig. 3 A, VVL) or when the second hydrophobic segment was absent (Fig. 3 A, 2). We imported Mgm11-228DHFR into isolated mitochondria with and without prior depletion of matrix ATP. Upon ATP depletion, generation of s-Mgm11-228DHFR was strongly reduced (Fig. 3 B). Finally, formation of s-Mgm11-228DHFR was strongly affected when isolated mitochondria derived from the ssc1-3 mutant were preincubated at the nonpermissive temperature before import experiments (Fig. 3 C). Therefore, the formation of s-Mgm11-228DHFR but not of l-Mgm11-228DHFR is ATP dependent, which most likely results from the ATP dependency of Ssc1. We suggest that the cleavage site for Pcp1 only becomes accessible and cleaved in the inner membrane when sufficient matrix ATP is present.
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Model of alternative topogenesis
Our data support a novel mechanism that regulates the balanced formation of both Mgm1 isoforms (Fig. 4). The mitochondrial membrane potential (Fig. 4, ) is sufficient to import the presequence of Mgm1 (residues 180) even at low levels of matrix ATP. The immediately following first hydrophobic segment can act as a stop-transfer sequence as shown previously for other preproteins (Neupert and Brunner, 2002). The efficiency of the stop transfer depends on the hydrophobicity of this segment. Processing by the mitochondrial processing peptidase and lateral insertion into the inner membrane lead to l-Mgm1. At high levels of matrix ATP the mitochondrial import motor "pulls in" part of the preprotein further and the second hydrophobic segment reaches the inner membrane. Pcp1 cleavage within this segment generates s-Mgm1. In this way, lateral insertion of the first hydrophobic segment into the inner membrane yielding l-Mgm1 and further ATP driven import with subsequent processing yielding s-Mgm1 are competing processes. This novel pathway of alternative topogenesis of Mgm1 during import into mitochondria is a key regulatory mechanism, which is crucial for the balanced formation of both isoforms. The process of alternative topogenesis implies that once its topology is established l-Mgm1 cannot be cleaved by Pcp1 because the cleavage site does not reach the protease in the inner membrane. Therefore, it is unlikely that the activity of Pcp1 is a physiologically important regulator of Mgm1 biogenesis. Consistent with this and in contrast to data by McQuibban et al. (2003), Pcp1 has not been found to be rate limiting for Mgm1 processing in our experiments (except when Pcp1 was down-regulated) at any growth stage including stationary cells (Fig. S1). Both isoforms are required for Mgm1 function (Herlan et al., 2003) and a strong shift in the ratio between both isoforms of Mgm1 is sufficient to alter mitochondrial morphology. We speculate that the ATP level in mitochondria, through alternative topogenesis, might play a role in controlling mitochondrial morphology. This would provide a molecular link between the bioenergetic state of mitochondria and their morphology. We hypothesize that mitochondrial damage such as the acquisition of mutations in mtDNA by oxidative stress would lead to reduced ATP levels in the matrix. Such damaged mitochondria may be prevented from fusing with intact mitochondria because formation of s-Mgm1 is impaired. Alternative topogenesis would serve as a mechanism that counterselects against bioenergetically disordered mitochondria and exclude them from the mitochondrial network and from inheriting the damaged mtDNA. A similar mechanism may apply to the human orthologue of Mgm1, OPA1, which is associated with the neurodegenerative disorder autosomal dominant optic atrophy type I (Alexander et al., 2000; Delettre et al., 2000). Therefore, alternative topogenesis of Mgm1/OPA1 may have major implications in the pathogenesis of mitochondrial diseases.
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Materials and methods |
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Fluorescence microscopy
Strains were cotransformed with plasmid pVT100U-mtGFP expressing mitochondria targeted GFP (Westermann and Neupert, 2000) and analyzed by standard fluorescence microscopy on an Axioplan 2 (Carl Zeiss MicroImaging, Inc.) with a NA 1.3 oil immersion objective (100x; model Plan-Neofluar; Carl Zeiss MicroImaging, Inc.) and a CCD camera 1.1.0 (Diagnostic Instruments) at RT using Metaview 3.6a software (Universal Imaging Corp.). The M28-82 mutant was stained with 0.1-µM rhodamine B hexyl ester (Molecular Probes). Classification of the morphology phenotypes was performed without knowledge of strain identity at the time of analysis.
In vitro import
In vitro import of radiolabeled precursor proteins was performed as described previously (Herlan et al., 2003). Matrix ATP was depleted by preincubation with 40 U/ml apyrase and 10 µg/ml oligomycin for 20 min at 25°C and subsequent addition of 5 µM atractyloside for 5 min at 4°C. After import mitochondria were treated with 50 µg/ml trypsin for 25 min at 4°C to remove proteins bound to the surface of mitochondria. Efficiency of ATP depletion and loss of Ssc1 function at 37°C in mitochondria isolated from the ssc1-3 strain were controlled by importing radiolabeled precursor of pSu91-69-DHFR, which is imported in an ATP- and Ssc1-dependent manner (Gambill et al., 1993).
Hydrophobicity analysis
Hydrophobicity plots were calculated according to Kyte and Doolittle (1982)(window size, 15) using ProtScale software (Swiss Institute of Bioinformatics on www.expasy.org).
Online supplemental material
Evidence that Pcp1 is not rate limiting for the processing of Mgm1 in stationary cells is provided in Fig. S1. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200403022/DC1.
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Acknowledgments |
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This work was supported by Deutsche Forschungsgemeinschaft, SFB 594, B8, Deutsches Humangenomprojekt/Nationales Genomforschungsnetzwerk (MITOP Project), and Fonds der Chemischen Industrie.
Submitted: 3 March 2004
Accepted: 23 March 2004
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References |
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