From the Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Mitochondrial biogenesis requires translocation
of numerous preproteins across both outer and inner membranes into the
matrix of the organelle. This translocation process requires a membrane potential () and ATP. We have recently demonstrated that the efficient import of a urea-denatured preprotein into the matrix requires GTP hydrolysis (Sepuri, N. B. V., Schülke, N.,
and Pain, D. (1998) J. Biol. Chem. 273, 1420-1424).
We now demonstrate that GTP is generally required for efficient import
of various preproteins, both native and urea-denatured. The GTP
participation is localized to a particular stage in the protein import
process. In the presence of
but no added nucleoside
triphosphates, the transmembrane movement of preproteins proceeds only
to a point early in their translocation across the inner membrane. The
completion of translocation into the matrix is independent of
but is dependent on a GTP-mediated "push." This push is likely
mediated by a membrane-bound GTPase on the cis side of the
inner membrane. This conclusion is based on two observations: (i) GTP
does not readily cross the inner membrane barrier and hence, primarily
acts outside the inner membrane to stimulate import, and (ii) the
GTP-dependent stage of import does not require soluble
constituents of the intermembrane space and can be observed in isolated
mitoplasts. Efficient import into the matrix, however, is achieved only
through the coordinated action of a cis
GTP-dependent push and a trans
ATP-dependent "pull."
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INTRODUCTION |
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Most mitochondrial proteins are synthesized as precursors in the cytosol and subsequently translocated into the organelle. Proteins targeted to various mitochondrial subcompartments (outer membrane, intermembrane space, inner membrane or matrix) follow a variety of import pathways that differ in their energy requirements. For example, a membrane potential is required for the translocation of a preprotein across the inner membrane, but not into or across the outer membrane (1-3). Translocation of proteins across both membranes into the matrix of the organelle is particularly complex. It requires the coordinated action of two separate translocases, one (Tom) located in the mitochondrial outer membrane and the other (Tim) located in the inner membrane (4-6). Also required are ATP-dependent interactions with molecular chaperones both outside and inside the organelle (for review, see Refs. 7 and 8).
Among the cytosolic chaperones are Hsp70 and mitochondrial import stimulating factor (MSF).1 Hsp70 generally interacts with preproteins to keep them in a translocation-competent conformation. MSF, on the other hand, specifically binds mitochondrial signal sequences, preventing and/or reversing preprotein aggregation, then targeting the precursors to the outer membrane. The MSF-dependent import pathway requires cytosolic ATP, whereas the Hsp70-dependent pathway does not (7). The import of urea-denatured precursors does not require added cytosolic chaperones and may or may not require ATP (9-12).
Matrix-localized Hsp70 (mt-Hsp70) interacts with the incoming polypeptide chain as it emerges on the matrix (trans) side of the inner membrane channel. This interaction prevents backward movement of the translocating protein. The two proposed models, "Brownian ratchet" (13, 14) and "molecular motor" (15, 16), posit that mt-Hsp70 and ATP-hydrolysis in the matrix participate in the vectorial movement of preproteins into this compartment. Mechanistic details of how mt-Hsp70 participates, however, remain unclear (for review, see Ref. 8).
Do nucleoside triphosphates (NTPs) other than ATP participate in
mitochondrial protein import? Specifically, do they contribute to the
driving force required for unidirectional translocation across the
mitochondrial membranes? More than a decade ago, GTP was found to be
capable of replacing ATP in promoting mitochondrial protein import (2,
17-19). Subsequent studies with ATP-depleted mitochondria, however,
concluded that GTP must first be converted to ATP to support import of
a hybrid precursor, pCOXIV-DHFR, into the matrix (20). It was argued
that other NTPs exert an effect only via their conversion to ATP (NTP + ADP NDP + ATP), presumably by nucleoside diphosphate kinase (NDP
kinase) located in the mitochondrial intermembrane space (IMS). The ATP
thus generated could then enter the matrix via the ADP/ATP carrier and
drive translocation. As these studies were performed using ATP-depleted
mitochondria, they could not rule out the possibility that GTP might
exert an effect in the presence of sufficient amounts of matrix ATP,
which is always required for import into the matrix (21).
We have recently demonstrated that the efficient import of urea-denatured pPut, the precursor of a soluble matrix protein of yeast mitochondria, is dependent upon GTP hydrolysis (22). In this report, we demonstrate that GTP significantly stimulates import of several authentic and chimeric precursors into the matrix. The GTP-mediated stimulation is independent of the structural integrity of the precursor protein to be imported; import of native as well as urea-denatured precursors is strongly stimulated by a direct participation of GTP. In intact mitochondria, proteins must cross both outer and inner membrane barriers to reach the matrix. However, when the mitochondrial outer membrane is disrupted by hypotonic shock to generate mitoplasts, translocation of proteins into the matrix can occur directly across the inner membrane (23, 24). We have determined the energy requirements during different stages of translocation of preproteins across mitochondrial membranes using both intact mitochondria and mitoplasts. The initial translocation of the N terminus of a precursor across the inner membrane requires a membrane potential but does not require the addition of ATP or GTP. It is the subsequent translocation of the remainder of the protein which is significantly stimulated by the addition of GTP. Efficient import into the matrix, however, is achieved through coordinated interplay of cis GTP- and trans ATP-dependent processes.
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EXPERIMENTAL PROCEDURES |
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Isolation of Mitochondria and Preparation of Mitoplasts-- Mitochondria were isolated from Saccharomyces cerevisiae strain D273-10B (ATCC24657) as described (25). To obtain mitoplasts (i.e. mitochondria with disrupted outer membrane), intact mitochondria (12.5 mg/ml in HSB (20 mM Hepes-KOH, pH 7.5, 0.6 M sorbitol, 0.1 mg/ml bovine serum albumin)) were subjected to hypotonic shock by diluting (1:5) with the same buffer lacking sorbitol. Following incubation on ice for 15 min, samples were centrifuged at 15,000 × g for 2 min at 4 °C. The mitoplast pellet was resuspended in HSB for subsequent use. A corresponding amount of the supernatant (IMS fraction) was added back in some mitoplast import assays as indicated (see Fig. 3D).
Expression of Precursors in Bacteria and Their Purification-- The genes encoding the precursor form of mitochondrial Hsp70 (pmt-Hsp70; Ref. 26) and Tim11 (27) were amplified from a yeast genomic library (Promega) by the polymerase chain reaction (PCR) using appropriate primers. The PCR products were subcloned into the NdeI and XhoI sites of pET21b (Novagen). The gene coding for the precursor of succinate dehydrogenase (pSDH) was amplified from the plasmid pNS15 (28). The resulting PCR product was digested with AseI and XhoI, and subcloned into pET21b that had been digested with NdeI and XhoI.
Escherichia coli--
BL21(DE3) cells carrying different
plasmids (pET21b/pmt-Hsp70, pET21b/Tim11, or pET21b/pSDH) were cultured
in M9 medium supplemented with 0.1 mg/ml ampicillin at 37 °C. When
cultures had reached an A600 of 0.8, expression
of the protein was induced by the addition of
isopropyl-1-thio--D-galactopyranoside to 1 mM. Following incubation for 15 min at 37 °C, rifampicin
(0.2 mg/ml) was added to inhibit transcription by the host RNA
polymerase. After an additional 15 min incubation at 37 °C, a
mixture of [35S]Met and [35S]Cys
(EXPRE35S35S, NEN Life Science Products) was
added to a final concentration of 0.05 mCi/ml and incubation was
continued for another 3 h at 37 °C. All three overexpressed
proteins were found to be sequestered in inclusion bodies. The
inclusion bodies containing pmt-Hsp70, pSDH, or Tim11 were solubilized
in 50 mM Tris-HCl, pH 7.5, containing 8 M urea
and purified on Ni-NTA resin (Qiagen). The final eluate was in 20 mM Hepes-KOH, pH 7.5, 0.4 M imidazole, 8 M urea. pPut was expressed and radiolabeled in bacteria and
subsequently purified as described (29).
Preparation of Native Preproteins-- The plasmid pSP64/pPut was linearized with DraI, and its transcription was carried out as described (25). The plasmid pGT/pCOXIV-DHFR was linearized with HindIII and transcribed using T7 RNA polymerase (30). pPut or pCOXIV-DHFR was synthesized in reticulocyte lysate (Promega) using the supplier's protocol. The post-ribosomal supernatant containing [35S]Met-labeled native preprotein (pPut or pCOXIV-DHFR) was dialyzed against 20 mM Hepes-KOH, pH 7.5, 40 mM KOAc, 10 mM Mg(OAc)2, and 1 mM dithiothreitol at 4 °C for 14 h. The initial sample volume was increased ~2.5-fold during dialysis. The dialysate was centrifuged at 15,000 × g for 15 min to remove any aggregates, and a small aliquot (2 µl) was used for each import reaction.
Import Assay-- Import reactions, consisting of mitochondria (100 µg of protein) or an equivalent amount of mitoplasts, were performed essentially as described (22). Unless otherwise indicated, isolated mitochondria or mitoplasts were used directly without manipulating the existing NTP levels. Urea-denatured precursors (40-50 ng/µl) were diluted 50-fold in the import reactions; the final urea concentration was 0.16 M. Following import, reaction mixtures were treated with trypsin (0.1-0.2 mg/ml) for 30 min on ice. To inactivate trypsin, samples were diluted with HSB containing 5 mg/ml soybean trypsin inhibitor, 100 units/ml Trasylol, and 1 mM phenylmethylsulfonyl fluoride. Mitochondria or mitoplasts were sedimented (15,000 × g for 10 min at 4 °C) and washed with 10% trichloroacetic acid. When trypsin treatment was omitted, import reaction mixtures were directly diluted with HSB containing protease inhibitor mixtures and subsequently processed as described above. Samples were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Bands representing the precursor (p) and mature (m) proteins were quantitated using the software NIH Image. The bands below mPut in several figures are likely due to partial cleavage of molecules in transit to the matrix. These lower Mr bands are not the result of incomplete digestion of nonimported molecules (22).
NTP Uptake Assay--
[-32P]ATP and
[
-32P]GTP, both with specific activity 25 Ci/mmol,
were purchased from ICN Biochemicals. Labeled NTPs were mixed with the
corresponding unlabeled NTP to obtain a stock of 5 µM with a specific activity of 2 Ci/mmol. NTP-uptake reactions (100 µl)
were performed with 50 µg of mitochondria in the import assay buffer
(i.e. HSB containing 40 mM KOAc, 10 mM Mg(OAc)2, 5 mM unlabeled Met,
and 1 mM dithiothreitol) essentially as described (31). Reactions were initiated by the addition of 5 µl of radioactive NTP
(5 µM, 2 Ci/mmol) and immediate mixing. Following
incubation at 30 °C for different time periods (10 s to 2 min),
mitochondria were sedimented (15,000 × g for 2 min at
4 °C). The resulting pellet was washed with HSB, and solubilized by
0.1 ml of 2% SDS. The total radioactivity was determined in a Beckman
LS 6000 liquid scintillation counter.
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RESULTS |
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GTP Is Generally Required for Efficient Import of Urea-denatured Preproteins into the Matrix-- We have recently shown that GTP plays a direct and essential role during efficient import of urea-denatured pPut into the matrix (22). To test whether GTP is more generally required for efficient translocation across or into the inner membrane, we tested the nucleotide requirements for import of three other purified and urea-denatured proteins: mt-Hsp70 (26), succinate dehydrogenase (SDH; Ref. 28), and Tim11 (27). The precursor forms of both mt-Hsp70 (pmt-Hsp70) and SDH (pSDH) contain a N-terminal signal sequence which, upon import, is removed by matrix-localized signal peptidase(s). Although mt-Hsp70 is a soluble matrix protein, SDH is peripherally attached to the matrix side of the inner membrane. On the other hand, Tim11 is an integral membrane protein of the inner membrane, and its precursor form does not appear to contain a cleavable signal sequence.
Mitochondria isolated from S. cerevisiae were used directly without manipulating the existing NTP levels. Import of urea-denatured pmt-Hsp70 into these mitochondria was carried out in the absence or presence of ATP or GTP for two different time periods (Fig. 1A). Samples were analyzed before and after trypsin treatment. When the membrane potential across the inner membrane was dissipated by valinomycin, no import was detected even in the presence of both ATP and GTP. There was neither any conversion of the precursor to the mature form (lane 5) nor any protection of the precursor from trypsin (lane 9). In the absence of added NTPs, a small portion of the precursor was converted to the corresponding mature form (lane 2), which was completely degraded by externally added trypsin (lane 6). This shows that endogenous mitochondrial NTPs are not enough to promote import. When ATP was added, the appearance of mature molecules was increased 2.5-fold, but they were still largely accessible to protease regardless of incubation time (compare lane 3 with 7, and lane 10 with 12). Compared with the ATP-containing samples, the appearance of mature molecules was further increased another ~2-fold in the presence of GTP (compare lane 3 with 4, and lane 10 with 11). More importantly, a significant portion of the total mature molecules that appeared upon incubation with GTP remained protected from trypsin (compare lane 4 with 8, and lane 11 with 13). The absolute GTP-mediated import efficiency after 6 min of incubation was ~40% (lane 13), whereas the corresponding ATP-mediated import efficiency was ~5% (lane 12). GTP hydrolysis was necessary as the import was strongly inhibited by the non-hydrolyzable (or slowly hydrolyzable) analog GTP
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Efficient Import of Native Preproteins into the Matrix Also Requires GTP-- We initially used urea-denatured precursors in our import assays in order to bypass any possible requirement for external ATP. However, urea-denatured precursors do not represent physiologic substrates in intact cells. Does the efficient import of a native preprotein into the matrix require GTP? To investigate this possibility (Fig. 2A, upper panel), pPut was synthesized in reticulocyte lysate cell-free translation system. Reticulocyte lysate is usually supplemented with ATP, an ATP-generating system, and GTP to support protein synthesis. A post-ribosomal supernatant containing the newly synthesized [35S]Met-labeled native pPut was therefore dialyzed extensively to remove free NTPs, NDPs, and other small molecules. This was then tested in the mitochondrial import assay. In the absence of added NTPs, about 25-30% of total pPut was converted to mPut in a membrane potential-dependent manner (data not shown). These mPut molecules, however, were not protected from externally added trypsin (lane 2), suggesting that although the signal sequence was cleaved by the matrix-localized signal peptidase, most of the C-terminal domain remained outside the organelle. This shows that residual NTPs still present in the dialysate were not sufficient to promote completion of import. Significant import was observed only when ATP or GTP was added in the import reaction. GTP-mediated import was 1.5-2-fold greater than that observed with ATP (lanes 3 and 4). This difference, however, was not as significant as that observed with urea-denatured precursors (Fig. 1, A and C; see also Ref. 22); this issue is addressed under "Discussion."
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Role of GTP in the Translocation of a Preprotein across the Inner Membrane into the Matrix-- Protein import across the outer and inner membranes of mitochondria into the matrix is a multistep process. GTP may regulate recognition and subsequent unfolding of precursors at the mitochondrial surface and/or provide energy for transmembrane movement of precursors into the organelle (22). The mitochondrial inner membrane contains a separate translocation system that is not permanently linked to that of the outer membrane (23, 24). We therefore tested the nucleotide requirements for import directly across the inner membrane into the matrix of mitoplasts.
Mitochondria were subjected to hypotonic shock, and the resulting mitoplasts were separated from soluble constituents of the IMS by centrifugation. More than 80% of endogenous cytochrome c peroxidase (IMS marker), but practically no endogenous Put (matrix marker), was detected in the supernatant (data not shown). These results indicate that hypotonic shock disrupted only the outer membrane, leaving most of the inner membrane intact. Import of native pPut (i.e. pPut synthesized in reticulocyte lysate and subsequently dialyzed) into mitoplasts was tested in the absence or presence of an ATP trap with or without addition of ATP or GTP (Fig. 3A). When no NTP was added, a significant portion of pPut was converted to mPut (lane 2). This conversion was unaffected by the presence of an ATP trap (compare lanes 2 and 8) but was completely abolished in the presence of valinomycin (data not shown). Furthermore, mPut molecules, which were generated in the absence of added NTPs, were completely degraded by externally added trypsin (lanes 5 and 11). These results suggest that the initiation of translocation across the inner membrane requires a membrane potential but does not require the addition of NTPs. When ATP was added, generation of mPut was increased 2-fold compared with a no NTP control (compare lanes 2 and 3). Still, only a very small fraction of mPut molecules was protected from trypsin (lane 6). As expected, the level of mPut production in the presence of both ATP and the ATP trap was comparable to that in the no NTP control (lanes 8 and 9), and this mPut was not protected from trypsin (lane 12). In contrast, the amount of mPut was 2-3-fold greater in the presence of GTP than with no added NTP, regardless of the presence of the ATP trap (compare lane 2 with 4, and lane 8 with 10). More importantly, the majority of mPut molecules that were generated in the presence of GTP remained protected from trypsin (lanes 4 and 7), and the presence of the ATP trap did not inhibit the GTP-mediated completion of import (compare lanes 7 and 13). The absolute GTP-mediated import efficiency in the absence or presence of the ATP trap was ~55-60% (lanes 7 and 13). These results suggest that GTP plays a critical role during import of native pPut across the inner membrane.
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Effects of Blocking the ADP/ATP Carrier on NTP-mediated
Import--
The complex issue of the interconversion of nucleotides,
presumed to occur through the action of an IMS-localized NDP kinase, perhaps could be definitively resolved by using a specific inhibitor of
the enzyme. To our knowledge, no such inhibitor is known. A gene
encoding the enzyme has been isolated from the yeast S. cerevisiae. The enzyme is not essential for cell viability, and
the yeast strains with the disrupted gene still possess some NDP kinase activity. Furthermore, the localization of the cloned gene product is
not clear (39). Likewise, no specific GTPase is commercially available.
This makes it difficult to directly study the conversion of ATP to GTP
during import. We therefore devised an alternative approach. Externally
added ATP can enter into the matrix via the ADP/ATP carrier. If ATP is
converted to GTP in the IMS compartment, one would expect that when the
ADP/ATP carrier is blocked more of the added ATP will be available to
be converted to GTP in the IMS, thereby stimulating import. We
therefore tested the import of urea-denatured mt-Hsp70 with different
concentrations of GTP or ATP when the ADP/ATP carrier was blocked with
CAT. As expected, blockage of the ADP/ATP carrier had no significant
effect on GTP-mediated import (Fig.
4A). In contrast, ATP-mediated
import was stimulated by 2-6-fold (Fig. 4B). Similar
results were obtained for the import of urea-denatured pPut (data not
shown). The CAT-conferred stimulation of ATP-mediated pPut import was
inhibited in a dose-dependent manner by GTPS (Fig.
4C). These results suggest that when the external
concentration of added ATP is kept high through the use of CAT, a
portion of it is converted to GTP, which then stimulates import.
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Comparison of the Inhibitory Effects of GTPS with That of GDP on
GTP-mediated Import--
Excess GDP could interfere with the GDP/GTP
exchange process of a GTPase cycle (40), thereby affecting the
GTP-mediated import process. To examine this possibility, the
GTP-mediated import of native pPut into mitoplasts was measured in the
presence of increasing concentrations of GDP or GTP
S. Import
reactions were carried out for 6 min at 30 °C (Fig.
5A). In the absence of GTP, no
import was detected with GTP
S (lane 8) or GDP (lane 9) alone. As expected, GTP
S inhibited GTP-mediated import in a
dose-dependent manner (lanes 3 and
4). However, under identical conditions GDP failed to
exhibit any inhibitory effect (lanes 5-7). These results
suggest that the inhibition of import by GTP
S is specific and not
due to any possible contamination of GDP associated with commercially
available GTP
S. This is an important consideration as commercially
available GTP
S could be contaminated with GDP by as much as
10%.
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GTP Acts Outside the Inner Membrane to Stimulate Import-- Although the ADP/ATP carrier of mitochondrial inner membrane has been extensively studied, no mitochondrial GDP/GTP carrier or translocator has so far been described. In fact, as in the case of the chloroplast inner envelope (41), the mitochondrial inner membrane appears to be impermeable to guanine nucleotides (42-45). This suggests that GTP stimulates import from outside the inner membrane. To validate this assumption, we examined the binding/uptake of radiolabeled GTP when incubated with isolated mitochondria under our import assay conditions. Radiolabeled ATP was used as a positive control (Fig. 6). As expected, ATP was rapidly taken up by mitochondria within 10 s. In contrast, depending on the incubation time (10 s to 2 min), the amount of radiolabeled GTP associated with mitochondria was only ~15-25% of that observed with ATP. The ATP-uptake was specific as it was strongly inhibited by CAT or ADP, but not by GDP. However, this assay could not distinguish between GTP bound to proteins outside the inner membrane and GTP actually taken up into the matrix. Our observations are consistent with the accepted notion that GTP is not readily taken up by mitochondria. Although we cannot formally exclude the possibility that over a long time period small amounts of GTP can cross the inner membrane barrier, the significant GTP-mediated import of urea-denatured precursors seen in short incubation times (1-3 min; Fig. 1A and Ref. 22) makes it reasonable to conclude that GTP added to the outside also acts on the outside of the inner membrane to stimulate import.
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Involvement of GTP in the Later Stages of Protein Import-- Earlier studies have demonstrated that an electrochemical potential is needed only for the early stages of import reactions (1). In our import assays using mitoplasts, a significant portion of native pPut was processed to mPut even in the absence of any added NTP. Although the signal sequence was cleaved by the matrix-localized signal peptidase, most of the C terminus of these mPut molecules remained exposed outside the inner membrane (Fig. 3A). The generation of these early intermediates was strictly dependent on a membrane potential (data not shown). These intermediates therefore provided a means of examining the nucleotide requirements for completion of transmembrane movement of a polypeptide chain through the inner membrane channel into the matrix.
Mitochondria were treated with CAT to block the ADP/ATP carrier and then subjected to hypotonic shock. Isolated mitoplasts were incubated with native pPut in the absence of added NTPs to generate early stage intermediates. About 55% of pPut was converted to early stage mPut intermediates (Fig. 7, lane 2) that were completely protease sensitive (lane 3). The ability of ATP, GTP or
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Both External GTP and Matrix ATP Are Required for Efficient
Import--
To delineate the individual roles of matrix ATP and
external GTP, matrix ATP was reversibly modulated as follows.
Efrapeptin, a potent inhibitor of the F1 moiety of
mitochondrial ATPase, was used to block respiration-driven ATP
synthesis. In addition, the passage of ATP and ADP across the inner
membrane was blocked by CAT. A combination of efrapeptin and CAT were
used to deplete free matrix ATP. When these ATP-depleted mitochondria
were supplemented with KG, matrix ATP was regenerated (21, 22,
35).
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DISCUSSION |
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We have demonstrated that GTP, or more specifically the hydrolysis
of GTP, is generally required for efficient import of
preproteins into the mitochondrial matrix. GTP plays a critical role in
the import of both native and unfolded preproteins. Furthermore, we have localized the GTP participation both temporally, i.e.
to a particular stage of the import process, and spatially,
i.e. to a particular portion of the import machinery. The
transmembrane movement of preproteins up to an early stage of
translocation across the inner membrane is dependent on a mitochondrial
membrane potential (), but does not require the addition of NTPs.
The efficient completion of protein translocation across the inner membrane into the matrix is independent of
but is dependent on a
GTP-mediated push (Fig. 7). Our data imply that a membrane-bound GTPase
on the cis side (outside) of the inner membrane participates in import across this membrane. Efficient import into the matrix, however, requires the coordinated interplay of a cis
GTP-dependent push and a trans
ATP-dependent pull.
Several observations demonstrate a direct role for GTP in mitochondrial protein import. (For all comparisons described below, import refers to the appearance of protease-protected mature molecules.) GTP-mediated import of purified, urea-denatured precursors into mitochondria or mitoplasts is 1.3-50-fold more efficient, depending on the experimental conditions, than that observed with an identical concentration of ATP (Figs. 1-4; see also Ref. 22). If the GTP-mediated stimulatory effect were dependent on a conversion of the GTP to ATP, then it is very unlikely that GTP could stimulate import to a higher level than that observed with an equivalent amount of ATP.
With preproteins synthesized in reticulocyte lysate, the difference
between GTP- and ATP-mediated import is much more significant in
mitoplasts (at least 8-fold, Fig. 3, A and B)
than in mitochondria (1.5-2-fold, Fig. 2A). This
discrepancy could arise from a reduced level of IMS components present
in the mitoplast preparation. It is possible that components present in
the IMS fraction, together with the numerous enzymes present in
reticulocyte lysate, facilitate the conversion of added ATP to GTP, and
thereby cause an indirect stimulation of preprotein import. This notion
is supported by the observation that the ATP-mediated import into
mitoplasts is stimulated by the addition of IMS fraction, and this
stimulation is strongly inhibited by GTPS (Fig. 3D).
Further evidence for the direct role of GTP in import is the persistence of its stimulatory effect in the presence of an external ATP trap (Figs. 2, 3A, and 7). Because of the rapid equilibration of NTPs across the mitochondrial outer membrane, any ATP potentially generated from GTP for action in the IMS should be consumed by this external ATP trap. The formal possibility exists that generated ATP could be utilized before its equilibration across the outer membrane, but the continued stimulatory effect of GTP on mitoplast import in the presence of the same ATP trap makes this possibility unlikely (Figs. 3A and 7).
How can we exclude the possibility that ATP, potentially generated from GTP in the IMS, avoids the ATP trap by rapidly entering the matrix via the ADP/ATP carrier, and then stimulates import from the matrix side of the inner membrane? This possibility is unlikely as GTP-mediated import remains practically unaffected when the ADP/ATP carrier is blocked with CAT, whether in the absence or presence of the ATP trap (Figs. 2B and 7). More importantly, we have demonstrated that matrix ATP alone is insufficient to stimulate import (Figs. 3B, 7, and 8).
What is the site of GTP action? Guanine nucleotides themselves appear
to be unable to penetrate the mitochondrial inner membrane (42-45).
Our observations support this notion by demonstrating that unlike ATP,
GTP is not readily taken up by mitochondria under the conditions of our
import assay (Fig. 6). It is therefore likely that GTP stimulates
import from outside the inner membrane. Furthermore, the efficiency of
GTP-mediated import in mitoplasts is comparable to that in intact
mitochondria, suggesting that soluble constituents of the IMS are not
required (Figs. 3C and D). These observations implicate the action of a membrane-bound GTPase on the cis
side of the inner membrane in the stimulatory effect. Support for this conclusion can be indirectly gleaned from the failure of a respiratory substrate (KG) alone to stimulate import (Figs. 3B, 7,
and 8). In the presence of
KG, substrate-level phosphorylation
results in the generation of matrix ATP via the tricarboxylic acid
cycle. During this process, GTP is also generated in the matrix as an intermediate (35). If GTP were to act from the matrix side of the inner
membrane,
KG alone should stimulate import to some extent.
A wide variety of conditions for protein import into mitochondria or
mitoplasts have been reported in the literature. These conditions vary
with respect to the nature and combination of components used as the
source of energy. Most import reaction mixtures, however, contained
ATP, an ATP-generating system, and respiratory substrates and NADH to
generate high matrix ATP and high membrane potential. Some, but not
all, also contained GTP. How efficient then is the GTP-mediated import
compared with that achieved under other widely used assay conditions?
In agreement with earlier reports, we have also observed efficient
import when both the matrix and external ATP concentrations are high,
i.e. in the presence of ATP, KG, and NADH (Fig.
3B). GTP alone, however, is able to drive import with
comparable efficiency, and the GTP-mediated import is only slightly
further stimulated by the combined addition of ATP,
KG, and NADH.
More importantly, the presence of an external ATP trap completely
abolishes the stimulation of import mediated by ATP,
KG, and NADH
combined, while having no effect on the stimulation mediated by GTP
alone (Fig. 3A) or a combination of GTP,
KG, and NADH
(Fig. 3B). These data do not contradict those previously
obtained with widely used assay systems, but rather pinpoint the
relative contribution of various components normally used as energy
source for mitochondrial protein import.
The individual roles of GTP and ATP are more clearly defined when the ATP concentrations both outside and inside the organelle are better controlled, i.e. when the ADP/ATP carrier is inhibited with CAT and the F1 moiety of mitochondrial ATPase is inhibited with efrapeptin to block respiration-driven ATP synthesis. In the presence of both efrapeptin and CAT, GTP-mediated import is strongly inhibited. This inhibition, however, is completely relieved by regenerating matrix ATP, whereas the addition of external ATP has no effect (Fig. 8). These results might explain why an earlier study failed to detect GTP-mediated import into ATP-depleted mitochondria (20). From these data, it is reasonable to conclude that efficient import into the matrix requires the coordinated interplay of a cis GTP-dependent push and a trans ATP-dependent pull. We suggest that for efficient import it is necessary, but not sufficient, that matrix ATP and external GTP each meets a threshold level. With this requirement met, any further elevation in the level of either of these components will result in a proportional increase in import efficiency toward its maximum. In this way, high levels of matrix ATP can compensate for low, but still threshold levels of external GTP, and vice versa. In this scenario, isolated mitochondria or mitoplasts contain threshold amounts of matrix ATP but not of external GTP. The addition of external GTP to this system thus allows efficient preprotein import to be achieved.
How do we explain efficient import mediated by a combination of ATP,
KG, and NADH in the absence of externally added GTP? It is possible
that a combination of high matrix and external ATP can bypass the GTP
requirement for efficient import. Alternatively, in the presence of
high concentrations of both matrix and external ATP, a portion of the
external ATP may be rapidly converted to GTP in the IMS, presumably by
NDP kinase. The GTP so generated meets the threshold requirement. This
notion is supported by the observation that added ATP is more efficient
at stimulating import when its external concentration is maintained at
a higher level through the use of CAT, and that the stimulatory
mechanism cannot bypass GTP-dependent processes (Fig.
4C). A high external ATP concentration may lead to a portion
of it being converted to GTP even in mitoplasts, with the aid of the
residual IMS fraction. This might explain why the import mediated by
the combination of ATP,
KG, and NADH was only slightly further
stimulated by GTP (Fig. 3B). The conversion of ATP to GTP in
mitoplasts is expected to be suboptimum, and the efficient import can
therefore be seen only in the presence of high concentrations of matrix
ATP generated by respiratory substrate(s) (Figs. 3 and 7).
An enzymatic conversion of ATP to GTP is expected to be temperature-dependent. Such an exchange process may be apparent in the kinetics of import at different temperatures. We have previously shown that the GTP-mediated import of urea-denatured pPut can occur to a significant extent even at lower temperatures (0-10 °C). In contrast, ATP can drive import to levels above 5% only at 30 °C. After a short incubation at 30 °C, GTP-mediated import reaches a plateau level, whereas ATP-mediated import is still in the early stage of the ascending linear range (22). With longer incubation (~20 min) at 30 °C, the difference between GTP- and ATP- mediated import is diminished (Fig. 3C). These results provide a possible explanation as to why some earlier studies, where import incubations were performed for longer durations at higher temperatures, failed to detect the GTP stimulatory effect over that mediated by ATP (22).
How does GTP stimulate mitochondrial protein import? We propose that the completion of protein translocation through the inner membrane channel into the matrix requires participation of a membrane-bound GTPase on the cis side of the inner membrane. The GTPase may transiently accompany the polypeptide chain in the mitochondrial inner membrane channel, as in the case of ATP-powered push by SecA in protein transport across the bacterial plasma membrane (for review, see Refs. 46 and 47). Alternatively, the GTPase may feed segments of the polypeptide chain into the inner membrane channel without actually penetrating the channel itself. The GTP-dependent push of the polypeptide chain across the inner membrane, in turn, may lead to unfolding of C-terminal domains that are yet to enter the organelle. The GTP requirement for efficient import, however, cannot be circumvented by altering the structural integrity of preproteins and/or by presenting them directly to the inner membrane in order to eliminate the need for translocation across the outer membrane.
An efficient unidirectional transmembrane movement of proteins across the inner membrane into the matrix is achieved only through the coordination of cis GTP- and trans ATP-dependent processes. Both of these processes are necessary; neither one can substitute for the other. The GTP-dependent push might be disengaged after mt-Hsp70 has sufficiently grabbed the incoming polypeptide chain. Alternatively, the process might continue until import is complete. The precise role of GTP in mitochondrial protein can be determined only after identification of a relevant GTPase(s).
GTP, shown here to be required for efficient import of proteins across the inner membrane into the matrix, may also participate in other stages of mitochondrial import, such as protein targeting. Indeed, GTP plays an important role in regulating protein targeting to the endoplasmic reticulum and chloroplasts (for review, see Refs. 48 and 49). Furthermore, as in the case of mitochondria, the outer and inner envelope membranes of chloroplasts contain independent protein translocation systems (50), protein import into chloroplast stroma requires stromal ATP (51), and the inner envelope membrane is impermeable to GTP (41). In light of the data presented here, it would be worthwhile to determine whether a similar GTP-dependent push and ATP-dependent pull is necessary for translocation of preproteins across the inner envelope membrane into the chloroplast stroma.
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ACKNOWLEDGEMENTS |
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The plasmid pGT/pCOXIV-DHFR was a gift from A. P. G. M. Van Loon. We thank Norbert Schülke for the plasmid pET21b/pSDH and for many helpful discussions. We also thank Andrew Dancis and Steve Cally for their valuable comments on the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the American Heart Association and the W. W. Smith Charitable Trust.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.
To whom correspondence and reprint requests should be addressed:
Dept. of Physiology, University of Pennsylvania School of Medicine,
D403 Richards Bldg., 3700 Hamilton Walk, Philadelphia, PA 19104-6085. Tel.: 215-573-7305; Fax: 215-573-5851; E-mail: pain{at}mail.med.upenn.edu.
The abbreviations used are:
MSF, mitochondrial
import stimulating factor; mt-Hsp70, matrix-localized 70-kDa heat shock
protein; IMS, intermembrane space; pCOXIV-DHFR, most of the signal
sequence of the precursor of yeast cytochrome c oxidase
subunit IV linked to dihydrofolate reductase Put, -1-pyrroline-5-carboxylate dehydrogenaseSDH, succinate
dehdrogenaseCAT, carboxyatractylosideGTP
S, guanosine
5'-3-O-(thio)triphosphate
KG,
-ketoglutarateHSB, Hepes-sorbitol-bovine serum albumin bufferPCR, polymerase chain
reactionm, maturep, precursor.
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REFERENCES |
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