From the Institut für Physiologische Chemie, Geethestrasse 33, 80336 München, Germany and § Centre de Genetique Moleculaire CNRS, Université Pierre et Marie Curie, 91190 Gif-sur-Yvette, France
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ABSTRACT |
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D-Lactate dehydrogenase
(D-LD) is located in the inner membrane of mitochondria. It
spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in
the intermembrane space. D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the
mitochondria in a -dependent, but
mt-Hsp70-independent manner. Upon import in vitro
D-LD folds in the intermembrane space to attain a
conformation indistinguishable from endogenous D-LD. Sorting of D-LD to the inner membrane is directed by a
composite topogenic signal consisting of the hydrophobic transmembrane
segment and a cluster of charged amino acids C-terminal to it. We
propose a model for the mode of operation of the sorting signal of
D-LD. This model also accounts for the driving force of
translocation across the outer membrane, in the apparent absence of
mt-Hsp70-dependent assisted import and involves the folding
of the D-LD in the intermembrane space.
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INTRODUCTION |
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Nuclear encoded proteins of the inner membrane of mitochondria
contain topogenic signals which function to sort them to the membrane
following their import. These topogenic signals comprise hydrophobic
cores of varying length and are flanked usually by charged amino acids.
In many instances these signals form integral parts, the transmembrane
anchors, of the sorted protein. In a small number of cases they are
proteolytically cleaved following the sorting event. These topogenic
signals are distinct from mitochondrial targeting sequences that serve
to target the precursor initially to the mitochondria and to initiate
membrane potential ()-dependent translocation across
the inner membrane. In some cases these topogenic signals are located
after N-terminal mitochondrial targeting signals (presequences) and
operate in conjunction with them. This is not the case for all
topogenic signals as many proteins of the inner membrane are
synthesized without presequences but rather contain internal
mitochondrial targeting signals.
Bearing these differences in mind, together with the wide variety of orientations displayed by inner membrane proteins, it would appear unlikely that the topogenic signals operate in an uniform manner. How do they act to ensure the sorting of proteins to the inner membrane? Recently we have described that the topogenic signals of a subset of inner membrane proteins serve as export signals directing the export of domains of the protein from the matrix to the intermembrane space following the complete import of the preprotein into the matrix (1-3). On the other hand, it has been suggested that some topogenic signals function as translocation arrest signals at the level of the inner membrane, thereby preventing further import into the matrix (4-8).
To further our understanding of the mechanisms of sorting of inner membrane proteins, we have addressed the biogenesis of the D(+)-lactate dehydrogenase (D-LD)1 protein. D-LD together with cytochrome b2, an L(+)-lactate dehydrogenase, are involved in lactate utilization in the yeast Saccharomyces cerevisiae and catalyze the oxidation of D- and L-lactate, respectively, to pyruvate. Cytochrome c acts as an electron acceptor in the oxidation reaction, connecting the reaction to the mitochondrial respiratory chain. Recently the gene for D-LD was cloned and appears to contain an N-terminal mitochondrial targeting signal (9).
The data presented here demonstrate that D-LD is a mitochondrial protein anchored to the inner membrane with an Nin-Cout orientation. We present here information on the topogenic signal sequence and energetic requirements necessary for D-LD to achieve this membrane orientation.
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EXPERIMENTAL PROCEDURES |
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Isolation of Yeast Mitochondria-- S. cerevisiae wild-type strain (D273-10B) was grown in lactate medium (10) at 30 °C, while the temperature-sensitive mutant of mt-Hsp70, ssc1-3 (PK83) and its corresponding wild type (PK82) (11) were grown at 24 °C. Cells were harvested at an A578 of ~1, and mitochondria were isolated, as described previously (10), with the exception that the zymolyase treatment of PK82 and PK83 strains was performed at 24 °C. Isolated mitochondria were resuspended in 0.6 M sorbitol, 20 mM HEPES, pH 7.4, 1 mM EDTA (SEH-buffer) at a protein concentration of 10 mg/ml.
Recombinant DNA Techniques and Plasmid Constructions-- The recombinant DNA techniques applied were as described by Sambrook et al. (12). The D-LD gene was obtained by amplification of yeast genomic DNA of strain D273-10B by a polymerase chain reaction. The resulting DNA fragment was cloned into pGEM4 (Promega, Madison, WI) yielding the plasmid pDLD. To construct the plasmids for the expression of pDLD(1-42)-dihydrofolate reductase (DHFR), pDLD(1-72)-DHFR, and pDLD(1-66+4)-DHFR, the relevant D-LD DNA fragments were synthesized by polymerase chain reaction and subcloned intermediate to a BamHI site in front of the DNA encoding mouse DHFR. Standard polymerase chain reaction and site-directed mutagenesis techniques were used to create the other pDLD(1-72)-DHFR derivatives used in this study.
Precursor Proteins-- DNA encoding precursor proteins were cloned in pGEM4 vectors and were transcribed with SP6 RNA polymerase. All precursor proteins were then synthesized in rabbit reticulocyte lysate (Promega) in the presence of [35S]methionine (13).
Import into Mitochondria-- Import was performed essentially as described previously in SI buffer (0.6 M sorbitol, 80 mM KCl, 50 mM HEPES pH 7.2, 3% (w/v) bovine serum albumin, 10 mM MgCl2, 2 mM potassium phosphate, 2.5 mM EDTA, 2.5 mM MnCl2) (14). Import mixtures contained 2 mM NADH, 2 mM ATP, 0.25 mg/ml mitochondrial protein/ml, and 1% (v/v) reticulocyte lysate containing the radiolabeled precursor proteins and were incubated at 25 °C, unless otherwise indicated. Following import for the time indicated, samples were either mock-treated or treated with proteinase K for 15 min at 0 °C either under nonswelling (0.6 M sorbitol, 20 mM HEPES, pH 7.4) or hypotonic swelling (20 mM HEPES, pH 7.4) conditions (14). Mitochondria and mitoplasts were reisolated by centrifugation, and samples were analyzed by SDS-PAGE and immunoblotting onto nitrocellulose. The efficiency of swelling of the mitochondria was assessed following immunodecoration of the blot with antisera against endogenous cytochrome c peroxidase (soluble intermembrane space protein) and Mge1p (matrix protein).
Depletion of Matrix ATP-- Isolated mitochondria were depleted of their free matrix ATP, as described previously (15). Briefly, following dilution in import buffer, mitochondria were incubated at 25 °C for 3 min, and then oligomycin (20 µM), carboxyatractyloside (5 µM), and NADH (2 mM) were successively added at 3-min intervals.
Assessment of the Folding State of D-LD-- Assessment of the folding state of imported and endogenous D-LD was performed as follows. Mitochondria were lysed with 0.1% (w/v) Triton X-100, 150 mM NaCl, and 10 mM Tris/Cl, pH 7.4, for 10 min on ice in the absence or in the presence of 7.5 µg/ml proteinase K or of 25 µg/ml trypsin. Proteins were then trichloroacetic acid-precipitated and analyzed by SDS-PAGE and Western blotting with an antibody directed against the C-terminal region of D-LD.
Antiserum Generation--
A BglII-HindIII
fragment encoding for the C-terminal residues (amino acids 373-576) of
D-LD was cloned into the Escherichia coli
expression vector pQE9 (digested BamHI-HindIII),
thus generating a (His6)-tagged D-LD C-terminal
fragment upon induction with
isopropyl-1-thio--D-galactopyranoside, which was
purified by Ni-nitrilotriacetic acid chromatography and used to
immunize rabbits to raise a polyclonal serum.
Miscellaneous-- Protein determination, SDS-PAGE, and Western blotting were performed according to the published methods of Bradford (16), Laemmli (17), and Towbin et al. (18), respectively. The detection of proteins after blotting onto nitrocellulose was performed using the ECL detection system (Amersham Corp.).
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RESULTS |
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Topology of the D-LD-- The amino acid sequence deduced from the cDNA sequence of the D-LD protein indicated the presence of an N-terminal targeting signal and a single transmembrane domain (residues 46-61, see Fig. 2A) (9). The predicted mitochondrial targeting signals comprises amino acids 1-25, thus leaving 20 residues prior to the transmembrane segment. The C-terminal hydrophilic segment comprises residues 62-576. To determine the submitochondrial localization of the D-LD, isolated mitochondria were subjected to protease treatment and subfractionation by hypotonic swelling, which disrupts the integrity of the outer membrane while leaving the inner membrane intact (Fig. 1A). D-LD was not accessible to externally added proteinase K in intact mitochondria. Upon hypotonic swelling, D-LD remained associated with the mitoplasts but was accessible to the added protease.
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Imported Radiolabeled Pre-D-LD Attains the Correct
Orientation in Vitro--
The open reading frame of D-LD
was cloned into a pGEM4 vector and transcribed with SP6 RNA polymerase.
Translation of the resulting mRNA in rabbit reticulocyte lysate in
the presence of [35S]methionine gave rise to a
radiolabeled product of approximately 63 kDa, termed
pre-D-LD (Fig. 1C). Upon incubation with
isolated mitochondria, pre-D-LD was imported into a
protease-resistant location where it was processed to its mature size
form of 61 kDa, termed m-D-LD. The processing is apparently
catalyzed by the divalent cation-dependent mitochondrial
processing peptidase (MPP), as it was blocked if mitochondria were
incubated with EDTA/o-phenanthroline prior to import.
Furthermore, a potential MPP cleavage site in the presequence of
pre-D-LD is observed after amino acid residue 25 (RYA), which is in agreement with the observed molecular
weight shift.
Signal Requirements for Sorting of D-LD-- To test whether the N-terminal region including the transmembrane region (amino acid residues 46-61) of pre-D-LD (Fig. 2A) contains all the necessary information to ensure correct sorting of the preprotein to an Nin-Cout topology across the inner membrane, a series of fusion proteins comprised of N-terminal regions of D-LD and fused to the mouse cytosolic DHFR were created (Fig. 2B).
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Role of Hydrophobicity for the Sorting of D-LD to the Inner Membrane-- To analyze the role of hydrophobicity in the targeting signal of D-LD, a series of proteins were created in which the hydrophobic core of the transmembrane segment was lengthened at its C-terminal end by the introduction of either 4 or 8 leucine additional residues. These modifications were performed either in pre-D-LD-(1-72)-DHFR and thus in the context of the cluster of charges or in the pre-D-LD-(1-66+4)-DHFR, i.e. in the absence of the charged cluster (Fig. 4A).
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Matrix ATP and mt-Hsp70 Requirements for Import and Sorting of D-LD-- To address whether the import of pre-D-LD is supported by the ATP-dependent mt-Hsp70 action in a similar manner as matrix-targeted proteins, we tested if depletion of matrix ATP levels had an adverse effect on the import and sorting of the process. Treatment of mitochondria prior to import with oligomycin and carboxyatractyloside serves to reduce the matrix levels so they become limiting for mt-Hsp70 action (15, 19). The efficiency of import of the pre-D-LD was only slightly reduced when imported into ATP-depleted mitochondria, as compared with matrix ATP-containing mitochondria (Fig. 5A). This was in marked contrast to the import of a hydrophilic matrix targeted protein, preSu9(1-69)-DHFR, whose processing by MPP and import were significantly reduced by the depletion of the matrix ATP levels. Likewise the import and the sorting of pre-D-LD(1-72)-DHFR was not affected by prior matrix-ATP depletion. In contrast, the import, but not the processing by MPP, of the matrix mistargeted pre-D-LD(1-66+4)-DHFR, was inhibited (Fig. 5B).
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Imported D-LD Folds in the Intermembrane Space to Its Native Conformation-- As shown in Fig. 1, endogenous D-LD contains a large C-terminal folded domain in the intermembrane space. When mitochondria were solubilized with detergent and subjected to a proteinase K treatment, a C-terminal protease resistant fragment (approximately 50 kDa) of D-LD was generated, as indicated by immunodecoration of the blots with an antiserum specific to the C-terminal region of D-LD (Fig. 6A). When the lysis was performed in the presence of trypsin, a single slightly larger C-terminal fragment was recovered (Fig. 6A), which according to its size (56 kDa) corresponds to the complete intermembrane space domain of D-LD.
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DISCUSSION |
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The sequence information responsible for targeting D-LD to the mitochondria and for sorting into the inner membrane resides entirely in the N-terminal 72-amino acid residues of D-LD. As shown here this short region contains both a cleavable mitochondrial targeting signal, which is followed by the inner membrane topogenic signal. The topogenic signal apparently serves to arrest the D-LD in the inner membrane during import.
Dissection of the topogenic signal revealed it to be a composite one, comprising essential hydrophobic and hydrophilic features. The hydrophobic transmembrane region is essential for sorting D-LD to the inner membrane. Furthermore, our results suggest that the degree of hydrophobicity of this segment is crucial for sorting of D-LD to the inner membrane. If the length (and thereby the hydrophobicity) is increased, D-LD preferentially partitions into the outer membrane in such a manner that further translocation to the inner membrane is prevented. The hydrophobic transmembrane segment is flanked directly on both sides by a positively charged amino acid. These charges appear to play a minor role in the sorting process of D-LD. Deletion of the cluster of charged amino acids located C-terminally of the hydrophobic domain, however, led to a massive missorting of the protein to the matrix. The cluster of charges is therefore one of the two major determinants of the topogenic signal required to attain the Nin-Cout orientation of D-LD, the second one being the transmembrane domain. Deletion of one of these two determinants results in the efficient missorting of the protein into the matrix. The importance of these two features suggests a specific recognition of the topogenic signal which involves both a hydrophobic-hydrophobic interaction as well as a hydrophilic-hydrophilic one.
Hydrophobic interactions may actually occur at the level of the translocase of the inner membrane (TIM machinery), where the transmembrane segments of Tim17 or Tim23, components of the TIM complex, may display an affinity for hydrophobic segments of incoming preproteins, and thus serve to retard further translocation across the inner membrane (20). Alternatively, it is feasible that a channel of the translocase may open out to the lipid bilayer and thus the hydrophobic domain of incoming D-LD may preferentially partition into the membrane from the TIM machinery, thus aborting further translocation.
Not every hydrophobic topogenic signal, however, functions to arrest preproteins in the inner membrane. Indeed many are imported into the matrix where they serve as re-export signals targeting insertion into the inner membrane (1-3). Hence there must be a second control mechanism to distinguish whether a topogenic signal should be arrested or further translocated. We propose this may constitute the role of the second topogenic signal determinant, the cluster of charged amino acids located at the C-terminal side of the transmembrane segment. The importance of this cluster indicates a degree of protein:protein interaction may also be involved in the mode of operation of the sorting signal. How this cluster of charges functions for the sorting of D-LD remains a matter of speculation. Due to their positioning with respect the transmembrane anchor, these charged residues are probably recognized by (a) proteinaceous factor(s) exposed to the intermembrane space; hydrophilic domains of Tim17 or Tim23, which are exposed to the intermembrane space could conceivably be involved. Alternatively, one could imagine that the cluster of charged amino acids might function to slow down the translocation across the inner membrane and thus serve to enhance the partitioning of the hydrophobic domain into the lipid bilayer of the inner membrane.
A similar cluster of charged amino acids are found at the C-terminal side of the transmembrane domain of other inner membrane proteins with an Nin-Cout orientation, such as Sco1p, Sco2p, and CoxVa. The mechanism which D-LD uses to achieve its membrane orientation may therefore be common to these other membrane proteins. The transmembrane domains of all these proteins are relatively short (16-18 amino acids) and are flanked on each side by a positive charge, with the exception of CoxVa which has a negative charged residue at the C-terminal position. The importance of both the short transmembrane segment and of the charged cluster in sorting of these other inner membrane proteins remains to be studied. A similar charged cluster is not found in the sorting signals of other proteins, e.g. cytochrome b2 and cytochrome c1, where a stop-transfer mechanism of sorting is currently being debated, thus supporting the case for an alternative sorting mechanism of these proteins (4, 5, 21-23).
What is the driving force for the import of D-LD across the
outer membrane? Translocation of the N terminus of D-LD
across the inner membrane into the matrix occurs in response to the
and does not require mt-Hsp70 and matrix ATP. Import of the N terminus to the matrix is a rapid process even at lower temperature. The topogenic signal of D-LD appears to function in halting
import in the inner membrane. No evidence for the transient passage of part or all of D-LD through the matrix during its sorting
to the inner membrane was obtained. As we have demonstrated previously, the presence of a hydrophobic sorting signal in proximity to the N-terminal matrix-targeting signal of a preprotein can secure the
translocating polypeptide chain in the import channel in a manner that
does not require mt-Hsp70 activity (20). Completion of translocation of
C-terminal elements across the outer membrane thus occurs independently
of mt-Hsp70, and in the case of D-LD, appears to be the
rate-limiting step for import. Upon import across the outer membrane,
D-LD folds to attain a protease-resistant conformation. The
kinetics and temperature sensitivity of this folding step paralleled
those of import. The folding of translocated domains on the trans side
of the outer membrane may be the driving force for the completion of
translocation across the membrane.
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ACKNOWLEDGEMENT |
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We are grateful to Evelyne Shechter for assistance in the cloning of the D-LD derivatives.
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FOOTNOTES |
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* This work was supported by grants from the Sonderforschungsbereich 184, Teilprojekt B2, and Münchener Medizinische Wochenschrift (to R. A. S.) and the Association pour la Recherche sur le Cancer (to B. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a fellowship of the European Union (Biotechnology
Program).
¶ To whom correspondence should be addressed. Tel.: 49 89 5996 295; Fax.: 49 89 5996 270; E-mail: stuart{at}bio.med.uni-muenchen.de.
1 The abbreviations used are: LD, lactate dehydrogenase; DHFR, dihydrofolate reductase; PAGE, polyacrylamide gel electrophoresis; MPP, mitochondrial processing peptidase; TIM, translocase of the inner membrane.
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REFERENCES |
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