From the Adolf Butenandt Institut für
Physiologische Chemie, Ludwig-Maximilians-Universität
München, Goethestrasse 33, 80336 München, Germany and
¶ Department of Biological Sciences, University of Alberta,
Edmonton, Alberta, T6G 2E9 Canada
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ABSTRACT |
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TOM22 is an integral component of the preprotein
translocase of the mitochondrial outer membrane (TOM complex). The
protein is anchored to the lipid bilayer by a central trans-membrane
segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we
describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The
orientation of the protein is not influenced by the charges flanking
its trans-membrane segment, in contrast to observations regarding
proteins of other membranes. In vitro import studies
utilizing TOM22 preproteins harboring deletions or mutations in the
cytosolic domain revealed that the combination of the trans-membrane
segment and intermembrane space domain of TOM22 is not sufficient to
direct import into the outer membrane. In contrast, a short segment of
the cytosolic domain was found to be essential for the import and
assembly of TOM22. This sequence, a novel internal import signal for
the outer membrane, carries a net positive charge. A mutant TOM22 in
which the charge of the import signal was altered to 1 was imported less efficiently than the wild-type protein. Our data indicate that
TOM22 contains physically separate import and membrane anchor sequences.
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INTRODUCTION |
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The biogenesis of mitochondria requires products of both the
nuclear and mitochondrial genomes (1). The nuclear-encoded proteins are
synthesized in the cytoplasm and contain signals that direct them to
the organelle and to their specific intramitochondrial subcompartments
(2, 3). The targeting signals in matrix-destined preproteins are
cleavable, positively charged, amino-terminal presequences that have
the potential to form amphipathic -helices (4, 5). In cooperation
with internal topogenic signals (6), this type of targeting signal also
directs some proteins to the inner membrane and the intermembrane
space.
In contrast to the extensive data regarding amino-terminal presequences, there is limited information about the signals that direct proteins to the outer membrane, and, via presequence-independent mechanisms, to the inner membrane and the intermembrane space. In most cases, these signals are not cleaved after import, and may be located internally (2, 3). The mitochondrial outer membrane contains several proteins that are anchored in the lipid bilayer via a single trans-membrane segment, exposing domains to the cytosol and/or the intermembrane space (7, 8). The most thoroughly studied example is Saccharomyces cerevisiae Tom70,1 a protein that is anchored to the membrane by an amino-proximal signal anchor (9, 10) that also contains all of the essential targeting information (11, 12). In contrast, Bcl-2 of the mammalian mitochondrial outer membrane (13, 14) and Tom6 of the S. cerevisiae TOM complex contain carboxyl-terminal signal anchor sequences (15, 16). Tom6 harbors additional targeting information that does not overlap with the trans-membrane segment (17).
During import of Tom70 (9), Bcl-2 (18), and Tom6 (15), segments of less than 11 residues cross the outer membrane. To investigate the targeting signals in proteins which possess larger domains that must traverse the membrane, we have chosen to study TOM22 of the Neurospora crassa TOM complex. The amino-terminal 84 residues of TOM22 are exposed to the cytosol and the carboxyl-terminal domain of 49 amino acid residues is localized to the intermembrane space (19). Essential targeting and/or assembly information in TOM22 does not reside in the intermembrane space segment of the protein, because truncated TOM22 preproteins lacking this domain are efficiently imported into mitochondria (20-22). In the current study, we investigated the role of the cytosolic domain in the import of TOM22 into mitochondria.
In addition to targeting signals, trans-membrane proteins contain information which determines their orientation across the bilayer. For instance, the orientation of Tom70 can be reversed, in the absence of a membrane potential, when its first 10 residues are replaced by a strong matrix-targeting signal (23, 24). The distribution of charges on either side of the membrane anchor is responsible for the orientation of proteins of the bacterial inner membrane ("positive-inside rule") (25) and the membrane of the endoplasmic reticulum ("charge-difference rule") (26, 27). In both cases, the retention of a protein segment on the cis-side of the lipid bilayer correlates with the presence of positively charged residues on that side of the membrane anchor. The remainder of the protein is transported across the membrane. TOM22, with its internal membrane anchor, is an ideal model protein for the examination of the determinants of protein orientation across the mitochondrial outer membrane. The trans-membrane segment of TOM22 is flanked by a single positively charged residue on its cytosolic side and by two negatively charged residues on the intermembrane space side (19). Yeast Tom22 has a similar distribution of flanking charges, with a single charged residue on either side of the membrane anchor (28-30). We tested the importance of the charged residues flanking the TOM22 trans-membrane segment for correct insertion of the protein into the outer membrane.
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EXPERIMENTAL PROCEDURES |
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TOM22 Derivatives--
The TOM22 derivatives used in this study
were generated from the tom22 cDNA (19). Coding
sequences for the truncated proteins TOM223-17 and TOM22
2-55
(see Fig. 3) were generated by amplification of the desired portion of
the tom22 cDNA by a polymerase chain reaction. The
resulting products were cloned into pGEM4 or pGEM2 (Promega). The
tom22
2-55 construct included the 16 bp upstream of the
wild-type tom22 initiation codon to ensure efficient
in vitro transcription and translation. The codon for
residue four in this sequence was changed from CCC to CCT (both
encoding proline) to avoid potential problems in the polymerase chain
reaction. The tom22
3-17 coding sequence began with the
initiation codon and did not require further upstream sequence for
efficient expression.
Import Reactions and Protease Protection and Alkaline Extraction Assays-- The wild-type laboratory strain, 74A, of N. crassa was grown and maintained under standard conditions (32). Mitochondria were freshly isolated and used in standard import assays as described (20, 33-35). Where indicated, mitochondria or outer membrane vesicles (see below) were treated with trypsin (20-40 µg/ml, 15 min, 0 °C) prior to the import reactions (20, 36). Following the import reactions, the import of each TOM22 derivative was assessed by a protease protection assay (19, 35). To confirm that TOM22 proteins were integrated into the membrane, duplicate import reactions were subjected to an alkaline extraction assay (20).
In the experiments involving DHFR-TOM22(78-154), the import reactions contained either vesicles derived from purified outer membranes (OMV) (36), or large unilamellar vesicles prepared by the extrusion technique (LUVET) (37). The LUVET were prepared from individual lipids purchased from Sigma. OM-LUVET had the same composition as outer membranes from Neurospora mitochondria (38) (45% phosphatidylcholine, 29% phosphatidylethanolamine, 15% ergosterol, 8% phosphatidylinositol and 3% cardiolipin). PC/PE-LUVET were composed of 15% ergosterol, 52% phosphatidylcholine, and 33% phosphatidylethanolamine. The liposomes were reisolated after production, and at subsequent steps in the analysis, by centrifugation at 220,000 × g for 60 min at 2 °C. Import into OMV and LUVET was performed under standard conditions (36), with two alterations. First, each sample contained 10 µg of OMV protein or the corresponding amount of lipid (20 nmol of inorganic phosphate). Second, import into LUVET was carried out in EM buffer (1 mM EDTA, 10 mM MOPS, pH 7.5) to allow efficient reisolation of the LUVET after the reaction. The reactions were stopped by the addition of 500 µl of SEM buffer (220 mM sucrose, 1 mM EDTA, 10 mM MOPS, ph 7.5) for OMV samples or EM buffer for LUVET. Following import, an alkaline-extraction assay was performed (36).Miscellaneous Procedures-- The following published or manufacturers' procedures were used: standard DNA manipulations (39), SDS-PAGE utilizing high Tris urea gels (40); blotting of proteins onto nitrocellulose (Bio-Rad); quantitation of radiolabeled proteins on dried gels and nitrocellulose membranes with a Fuji BAS-1500 Bioimaging analyzer. mRNA was prepared in vitro as described previously (41) or with the RibomaxTM system (Promega). Radiolabeled preproteins were synthesized in vitro as described elsewhere (41), or with the TNT SP6-coupled reticulocyte lysate system (Promega), in the presence of [35S]methionine as a label.
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RESULTS |
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Charged Residues Flanking the Trans-membrane Segment of TOM22 Do Not Influence Its Import into, or Orientation across, the Outer Membrane-- The importance of the charged residues flanking the trans-membrane segment of TOM22 for its import and correct assembly in the outer membrane was assayed by utilizing a series of mutant TOM22 preproteins in which one or both of these sets of flanking residues was replaced by either neutral amino acids, or ones of the opposite charge (Fig. 1). Radiolabeled preproteins were imported into mitochondria and their assembly into the outer membrane was assayed by partial digestion with proteinase K (protease protection assay). The pattern of proteolytic fragments generated from each of the TOM22 mutants resembled that of wild-type TOM22 (Fig. 1), indicating that each of these proteins became imported in the correct orientation. All preproteins were imported with approximately the same efficiency, with the exception of TOM22R84D which was reduced in import to about one-third of the wild-type TOM22 level. Complete reversal of the flanking charges had no effect on the orientation in the membrane and reduced import to about two-thirds of the wild-type level. In all cases, the protease-protected fragments were absent, or present at much lower levels, when trypsin-pretreated mitochondria were employed in the import reactions (Fig. 1, +pre-Tryp). This demonstrates that these mutant preproteins, like wild-type TOM22 (20, 35), require the protease-sensitive components of the TOM complex for their import and are degraded by added protease if not assembled into the membrane. Therefore, the charges flanking the trans-membrane segment of TOM22 do not play an essential role in the orientation and assembly of this preprotein into the TOM complex.
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The Trans-membrane Segment of TOM22 Does Not Function as a Signal Anchor Sequence-- To determine whether the carboxyl-terminal portion of TOM22 functions as a mitochondrial signal anchor (11-14, 42), we analyzed the import of DHFR-TOM22(78-154), in which the passenger protein mouse DHFR replaces the amino-terminal cytosolic domain of TOM22. This fusion protein includes the trans-membrane segment and the intermembrane space domain of TOM22 (Fig. 2A). To avoid nonspecific integration of a signal anchor sequence into non-mitochondrial membranes that can contaminate normal mitochondrial preparations, the import reactions were performed using vesicles derived from highly purified outer membranes (OMV). The possibility of lipid-mediated insertion events was assayed in experiments utilizing artificial liposomes (LUVET) with the same lipid composition as mitochondrial outer membranes (OM-LUVET) (38), or LUVET lacking negatively charged lipids (PC/PE-LUVET). In all cases, import was assayed using the alkaline extraction method, which removes all proteins that are not integrated into the membranes. Wild-type TOM22 was imported efficiently into intact OMV (Fig. 2B) (36), but not into trypsin-pretreated OMV, OM-LUVET, or PC/PE-LUVET. Thus, in agreement with previous results using whole mitochondria (20, 35), the integration of TOM22 into the outer membrane is strictly dependent on proteinaceous surface receptors. In contrast, the amount of DHFR-TOM22(78-154) recovered with intact OMV, and all other membranes tested, was barely above the background levels found in the absence of membranes (Fig. 2B). We conclude that the carboxyl-terminal part of TOM22, comprising the trans-membrane segment and the intermembrane-space domain, is not sufficient to target a protein to the outer membrane. The targeting signal must therefore reside in the cytosolic domain.
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The Cytosolic Domain of TOM22 Contains an Internal, Positively
Charged Sequence Required for Import--
TOM22 preproteins harboring
various deletions in their cytosolic domains (Fig.
3) were utilized to investigate the role
of this region of the protein in targeting and assembly of TOM22. Radiolabeled mutant preproteins were incubated with isolated
mitochondria, and their import was assessed with the protease
protection assay. A fragmentation pattern related to that of the intact
TOM22 protein was generated, with similar efficiency, from
TOM223-17, TOM22
18-29, and TOM22
30-44 (Fig.
4A). Minor qualitative
differences in the patterns of fragments from the various mutants are
most likely due to the distinct segment missing in each protein. The
typical fragmentation patterns were not formed after import into
trypsin-pretreated mitochondria (+pre-Tryp), demonstrating
that these mutant proteins require protease-sensitive components
of the TOM complex for their import (20, 35). In contrast,
TOM22
45-59 and TOM22
61-75 (Fig. 3) were not imported into
mitochondria, as tested using both the protease protection assay
(Fig. 4, A and B) and the alkaline extraction method (not shown). TOM22
77-84 (Fig.
4B), which harbors a deletion adjacent to the trans-membrane
segment, was imported into mitochondria about 4-fold less efficiently
than the wild-type TOM22 (Fig. 4A). Together, these data
indicate that the region bounded by residues 45-75, or certain
residues within it, is essential for import, while residues between 77 and 84 enhance the efficiency of the process. We will refer to the
segment encompassing residues 45-75 as the "import sequence."
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Import of TOM22(105) Requires the Import Sequence in the Cytosolic
Domain--
TOM22(105) lacks the carboxyl-terminal, intermembrane
space domain and is efficiently imported via a mechanism that does not require the protease-sensitive receptors on the mitochondrial surface
(20). The identification of the import sequence in the cytosolic domain
of TOM22 allowed us to determine whether TOM22(105) is targeted to the
outer membrane through a mechanism involving this signal. If this is
the case, mutation of the import sequence in TOM22(105) should severely
inhibit its import. To examine this question, we assessed the import
competence of TOM22(105)61-75, which lacks both the
carboxyl-terminal domain and part of the import sequence (Fig. 3). Only
a small fraction of this preprotein (approximately 2% of input) was
imported into intact or trypsin-pretreated mitochondria (Fig.
4B). In contrast, TOM22(105) is efficiently imported
into the organelles (40-60% of input) (20). Thus, the import sequence
is also necessary for the efficient import of TOM22(105).
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DISCUSSION |
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TOM22, a bitopic mitochondrial outer membrane protein that exposes domains to both the cytosol and intermembrane space, contains an essential import sequence that is located in the cytosolic domain and encompasses residues 45-75. The segment containing residues 77-84 enhances the efficiency of import, but is not absolutely required for the process. The close proximity of the TOM22 import sequence and the trans-membrane segment are reminiscent of that of the corresponding sequences in the BCS1 protein of the mitochondrial inner membrane (44). A hairpin structure formed between these two segments in BCS1 may be required for the import of the protein. By analogy, a similar structure could be formed in TOM22.
The TOM22 import sequence resembles matrix-targeting presequences (4,
5) in that it is enriched in serine, tyrosine, and threonine residues
(a total of 11 out of 31), and is potentially amphipathic (45).
Furthermore, the TOM22 import sequence carries a net positive charge
which we have shown to be important for its function (Figs. 4 and 5).
However, the import characteristics of TOM22(105) (20) and
TOM22(105)61-75 (Fig. 4B) indicate that the TOM22 import
signal does not depend on the protease-sensitive receptors of the
mitochondrial surface for its function, in striking contrast to
matrix-targeting presequences (2, 3). TOM22(105), unlike TOM22, is
efficiently imported into mitochondria in the absence of the
protease-sensitive components of the TOM complex (20). Therefore,
assuming that TOM22(105) and TOM22 are imported along the same pathway,
the import sequence must be able to function in the absence of surface
receptors. To demonstrate that TOM22(105) requires the import sequence
for its import, we deleted part of this signal from TOM22(105) to
create TOM22(105)
61-75. This preprotein was not efficiently
assembled into mitochondria (Fig. 4B), indicating that the
import sequence is required for the import of TOM22(105), and therefore
can mediate this process without the participation of the surface
receptors.
With which components of the outer membrane does the TOM22 import sequence interact? The most likely candidates are the protease-resistant proteins of the TOM complex, such as the membrane-embedded TOM40 (46) (reviewed in Lill and Neupert (47)), or TOM5 (48). Both proteins are in close contact with preproteins in transit across the outer membrane and are likely involved in protein transfer through the import pore. Interactions with the portions of the protease-sensitive TOM proteins that remain after tryptic digestion, or with outer membrane lipids, could also contribute to import.
What is the role of the protease-sensitive components of the TOM complex in the import of wild-type TOM22? The requirement for these receptors in the import of TOM22 (35) and some of its amino-terminal deletion derivatives (Fig. 4), but not of TOM22(105) (20), indicates that this dependence is imparted by the carboxyl-terminal domain of the protein. Protease-sensitive receptors are required for efficient unfolding and translocation of other preproteins (49). Thus, it is conceivable that interactions between TOM22 and the receptors position the carboxyl-terminus of TOM22 close to the translocation site and/or promote unfolding of this domain to facilitate its translocation through the import pore. Alternatively, interactions between the carboxyl-terminal domain and the receptors might be required to expose the import sequence for subsequent steps along the import pathway; this interaction would not be necessary in proteins such as TOM22(105) that lack the carboxyl-terminal domain.
Interactions of TOM22 with components on the surface of the outer membrane could also determine the ultimate orientation of the protein, which was not influenced by alterations to the charge distribution across the trans-membrane segment (Fig. 1). A strong interaction between the TOM22 import sequence and the TOM complex could prevent translocation of the amino-terminal domain across the membrane. As such, the TOM22 import sequence would functionally resemble the amino-terminal "retention signals" created and analyzed by Shore and co-workers (23, 24) in their studies of Tom70. An alternative hypothesis (see 8), that a tightly folded cytosolic domain may not be amenable to translocation, seems unlikely for TOM22 because deletion of various segments of the amino-terminal domain had no effect on orientation. Finally, the preponderance of acidic residues in the amino-terminal domain could conceivably prevent its insertion into the negatively-charged outer membrane. This possibility is unlikely because deletion (Fig. 4) or replacement2 of many of these negative charges does not influence the final orientation of the protein in the outer membrane.
TOM22 appears to be partitioned into functional units. The negatively charged, amino-terminal region is not required for import of TOM22 into the membrane. This domain appeares to be required for the proper function of TOM22 in the translocase complex (50), although the negative charges are not essential for this function.2 The positively charged portion of the cytosolic domain proximal to the trans-membrane segment harbors information essential for the import of TOM22 itself; the functional role of this domain in the import of other preproteins remains to be assessed. The trans-membrane segment anchors the protein to the outer membrane and is most likely involved in the formation of the import channel, as bypass import (43) does not occur in mitochondria depleted of TOM22 (34). Finally, the intermembrane space domain plays a role in the later stages of translocation across the outer membrane and in preprotein transfer to the inner membrane (20-22, 50).
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ACKNOWLEDGEMENTS |
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The expert technical assistance of M. Braun, B. Crowther, and P. Heckmeyer is appreciated. We thank Dr. Rosa Esteban, Instituto de Microbiologia-Bioquimica, Salamanca, Spain, for providing general laboratory facilities and supplies used in portions of this work.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada (MRC) (to F. E. N.), the Natural Sciences and Engineering Research Council (to D. A. C.), and the Sonderforschungsbereich 184 of the Deutsche Forschungsgemeinschaft (to R. L. and W. N.).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.
§ Recipient of a postdoctoral fellowship from the European Molecular Biology Organization. Permanent address: Instituto de Microbiologia Bioquimica, Departamento de Microbiologia y Genetica, CSIC/Universidad de Salamanca, 37000 Salamanca, Spain.
Supported through European Union Training and Mobility in
Research Grant 961229.
** Permanent address: Institut für Zytobiologie, Philipps Universität Marburg, Robert-Koch-Str. 5, 35033 Marburg, Germany.
Recipient of a postdoctoral fellowship from the Medical
Research Council of Canada. To whom correspondence should be addressed: Dept. of Microbiology, University of Manitoba, Winnipeg, Manitoba, R3T
2N2 Canada. Tel.: 204-474-8473; Fax: 204-474-7603; E-mail: Deborah_Court{at}umanitoba.ca.
1 The abbreviations used are: TOM, translocase of the mitochondrial outer membrane; TOMX and TomX, X-kDa protein of the Neurospora crassa and Saccharomyces cerevisiae TOM complexes, respectively; DHFR, dihydrofolate reductase; LUVET, large unilamellar vesicles prepared by extrusion technique; OMV, outer membrane vesicle; MOPS, 4-morpholinepropanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PC, phosphatidylcholine; PE, phosphatidylethanolamine.
2 Nargang, F. E., Rapaport, D., Ritzel, R. G., Neupert, W., and Lill, R. (1998) Mol. Cell. Biol., in press.
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REFERENCES |
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