©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Import of Transcription Factor MTF1 into the Yeast Mitochondria Takes Place through an Unusual Pathway (*)

Arunik Sanyal (2), Godfrey S. Getz (2) (1) (3)(§)

From the (1) Departments of Pathology, (2) Medicine, and (3) Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We report studies of the import into mitochondria of MTF1, a nucleus-encoded factor that markedly stimulates the specific transcription of mitochondrial DNA. Unlike most of the mitochondrial precursor proteins studied so far, import of MTF1 does not appear to require a receptor on the outer membrane, membrane potential across the inner membrane, or ATP hydrolysis. Its import is not affected by low temperature. It lacks a cleavable presequence but translocates across the inner membrane through its amino terminus; its sorting is independent of hsp60. Our results indicate an unusual and distinct import pathway for MTF1 into the yeast mitochondria.


INTRODUCTION

Mitochondria have their own genome (1) , which is transcribed by a machinery that consists of a core polymerase (2) and a mitochondrial transcription factor MTF1 (3) , both of which are encoded in the nucleus and are imported into the mitochondria from the cytoplasm. MTF1 is a 43-kDa protein required for specific mitochondrial transcription. Its predicted amino acid sequence as specified by its gene and the amino-terminal amino acid sequence of the mature protein purified from isolated mitochondria indicates that following translocation, only the initiating methionine is removed in transit to the mitochondria (4) .

There are several steps involved in the import of proteins into mitochondria and their proper sorting within mitochondrial subcompartments. These include binding of the precursor protein to a receptor on the outer membrane; unfolding of the precursor protein into a translocation competent conformation; translocation through contact sites between the outer and the inner membranes, which is dependent on the electrochemical potential across the inner membrane. During or after translocation, the targeting sequence of the precursor is cleaved by matrix-localized proteases to generate mature protein, which is then refolded and sorted to its proper submitochondrial location and in many cases is assembled into multimeric complexes (5, 6) . In some cases, the targeting sequence is included within the mature protein sequence so that following import there is no reduction in the size of the imported protein.

In this paper, we report that the import of MTF1, a nucleus-encoded mitochondrial protein, takes place through an unusual pathway, which does not require the receptor on the outer mitochondrial membrane, the membrane potential across the inner membrane, and ATP hydrolysis. Its sorting is independent of hsp60, although it moves across the inner membrane.


EXPERIMENTAL PROCEDURES

Materials

Carbonyl cyanide m-chlorophenylhydrazone (CCCP),() valinomycin, and apyrase were purchased from Sigma. Digitonin was purchased from ICN Pharmaceuticals Inc. The vector pGEM3 and rabbit reticulocyte lysate were purchased from Promega (Madison, WI). [S]Methionine (specific activity, 800 Ci/mmol) was purchased from Amersham Corp.

Isolation of Mitochondria

Mitochondria were isolated from the yeast strain D-273-10B (ATCC 25657) according to the method of Daum et al.(7) .

Isolation of MTF1 Gene by Polymerase Chain Reaction

The whole coding sequence of the MTF1 gene was isolated from yeast genomic DNA, prepared from the yeast strain D-273-10B according to the method of Cryer et al.(8) , by the polymerase chain reaction using two oligonucleotides chosen based upon the published sequence (3) .

In Vitro Coupled Transcription and Translation

The coding sequence of the MTF1 gene was placed under the control of the T promoter of the pGEM3 vector. The gene was transcribed and translated in vitro as follows. Three µg of the linearized plasmid DNA was transcribed in a 25-µl reaction mixture containing 40 mM Tris-HCl, pH 7.5, 6 mM MgCl, 2 mM spermidine, 10 mM NaCl, 80 units of RNasin, 10 mM dithiothreitol, 500 µM each of ATP, CTP, UTP, 80 µM GTP, and 500 µM mG(5`)ppp(5`)G using 40 units of T RNA polymerase at 37 °C for 30 min. The transcription reaction was stopped by adding 11 units of DNase I followed by incubation at 37 °C for 10 min. One µl of this transcription reaction mixture was used as a source of capped mRNA in a 25-µl translation mix that contained 17.5 µl of rabbit reticulocyte lysate, 20 units of RNasin, 0.5 µl of 1 mM amino acid mixture minus methoinine, 20 µCi of [S]methionine. The reaction mixture was incubated at 30 °C for 2 h. The lysate containing the labeled synthesized protein was then used for the in vitro import assay. The same methodology was employed for the coupled transcription/translation of control genes viz. F and ADP/ATP carrier (AAC).

In Vitro Import

Import of the in vitro synthesized protein into the isolated mitochondria was carried out according to the method of Cheng et al.(9) . The import buffer contained 0.6 M sorbitol, 3% bovine serum albumin, 10 mM MOPS, pH 7.2, 80 mM KCl, 2 mM ATP, and 1 mM NADH.

Pretreatment of Mitochondria with Trypsin

A mitochondrial suspension (500 µl) containing 1 mg of mitochondrial protein was incubated with 30 µg of trypsin at 25 °C for 15 min. 10-fold excess of soybean trypsin inhibitor and 0.2 mM PMSF were then added and incubated at 25 °C for 3 min and at 4 °C for 10 min. The mitochondria were recovered by centrifugation in a microcentrifuge; washed once with a buffer containing 0.6 M sorbitol, 10 mM Tris, pH 7.5, 0.1 mg/ml soybean trypsin inhibitor, plus 0.1 mM PMSF; suspended in a small volume of the above mentioned buffer; and used for import after determination of the protein concentration.

Treatment of Mitochondria and the Rabbit Reticulocyte Lysate with Apyrase

150 µg of mitochondrial protein was suspended in 200 µl of buffer containing 0.6 M sorbitol, 3% bovine serum albumin, 10 mM MOPS, pH 7.2, 80 mM KCl, and 5 mM MgCl, incubated with 5 units of apyrase at 25 °C for 15 min, and reisolated by centrifugation in a microcentrifuge. Forty µl of rabbit reticulocyte lysate, containing the labeled synthesized protein, was diluted 5-fold with a buffer that contained 0.6 M sorbitol, 10 mM MOPS, pH 7.2, 80 mM KCl and incubated with 5 units of apyrase at 25 °C for 15 min.

Preparation of Mitoplasts

Mitochondria (1 mg of protein) prepared as stated above, was resuspended in 300 µl of buffer containing 0.1 M sorbitol, 10 mM Tris, pH 7.5, and incubated on ice for 30 min. Mitoplasts were recovered by centrifugation in a microcentrifuge and resuspended in a buffer containing 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5, and the protein concentration was measured.

Digitonin Treatment of Mitochondria

Mitochondria, after import of the precursor protein, were reisolated and washed twice with a buffer containing 0.6 M sorbitol, 10 mM Tris-HCl, pH 7.5, resuspended in 100 µl of the same buffer containing either 0.25 or 0.5% of digitonin, and placed on ice for 3 min. 500 µl of the above-mentioned buffer was added, followed by centrifugation in a microcentrifuge at 4 °C. One set of pellets was suspended in 100 µl of import buffer (3% bovine serum albumin, 0.6 M sorbitol, 10 mM MOPS, pH 7.2, 80 mM KCl) and incubated with 40 µg of proteinase K on ice for 15 min followed by the addition of 1 mM PMSF.

Estimation of Protein

The protein was estimated according to the method of Lowry et al.(10) .

RESULTS

MTF1 Can Be Imported into the Isolated Mitochondria

Many nucleus-encoded mitochondrial proteins are synthesized with amino-terminal presequences for targeting to the mitochondria. Usually the presequences are 20-80 amino acids long and contain several basic and hydroxylated residues and no acidic residues. However, it is still not clear which structural motif or sequence determines the targeting function (6) . MTF1, a nucleus-encoded mitochondrial protein, contains an aspartic acid at amino acid position 10 from the amino terminus, and the first 10 amino acids contain only 1 basic residue (lysine) and 1 hydroxylated residue (serine) (3) , indicating either a very short or no cleavable presequence. The amino-terminal amino acid sequence of the 43-kDa protein is reported to be identical with that specified by the MTF1 gene, except for the removal of the initiating methionine (4) . We have investigated the import of the MTF1 gene product into isolated mitochondria. The MTF1 gene in the pGEM3 vector was employed for coupled trasnscription and translation (as described under ``Experimental Procedures''), and the resulting labeled protein was used for the assay of import into the mitochondria isolated from Saccharomyces cerevisiae. The results are illustrated in Fig. 1 . Lane1 shows no product in the absence of the added MTF1 mRNA. When the in vitro synthesized MTF1 mRNA was added, a 42.6-kDa protein was obtained (lane2). This protein was found to be associated with the mitochondria after incubation at 25 °C (lane3), but no reduction in size was observed. The protein was resistant to added proteinase K (lane4). The mitochondria were reisolated after import and lysed with Nonidet P-40 when the protein was sensitive to proteinase K (lane5), indicating translocation of labeled MTF1 inside the mitochondria. The imported band could be immunoprecipitated by polyclonal antiMTF1 antiserum (lane7), raised against recombinant MTF1 overexpressed in E. coli,() but not by preimmune serum (lane6) or by antiCEM1 antiserum (lane8), an unrelated antibody raised against the CEM1 gene (12) .


Figure 1: In vitro synthesis and import of MTF1 into the isolated mitochondria. In vitro synthesized mRNA of MTF1 gene was used to synthesize the protein in rabbit reticulocyte lysate in the presence of [S]methionine (as described under ``Experimental Procedures''), and the labeled protein was then used for the import into the mitochondria isolated from D-273-10B cells. Lane1, 10 µl of the translation mix without mRNA; lane2, 10 µl of the reaction mix with mRNA; lane3, 20 µl of the translation mix containing the labeled protein was incubated with 75 µg of mitochondrial protein in a 100 µl of import reaction buffer at 25 °C for 30 min; lane4, same as in lane3 and after import 40 µg of proteinase K was added to the import reaction mix and incubated on ice for 30 min followed by the addition of 1 mM PMSF; lane5, same as in lane3 and after import the mitochondria were reisolated and disrupted with 1% Nonidet P-40 and 80 mM KCl followed by digestion with 40 µg of proteinase K; lane6, same as in lane4 and after the addition of PMSF the mitochondria were reisolated and suspended in 50 µl of 3% SDS, boiled for 3 min, and diluted 14-fold with a buffer (final concentration becomes 50 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 1 mM EDTA). 70 µl of preimmune serum was then added, and the whole mixture was rotated end-over-end overnight. 50 µl of protein A-Sepharose CL4B suspension in the above-mentioned buffer was added and rotated for 12 h. Protein A-Sepharose beads were recovered by centrifugation in the microcentrifuge, washed 3 times in the above-mentioned buffer, and then boiled with Laemmli gel loading dye. Lane7, same as in lane 6 but 70 µl of anti-MTF1 antiserum was used instead of preimmune serum; lane8, same as in lane6 and instead of preimmune serum, 70 µl of antiCEM1 antiserum was added. All of the samples were then analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.



Protease-digested Mitochondria Are Capable of Importing MTF1

Functional studies with Neurosporacrassa and yeast have suggested the presence on the mitochondrial outer membrane of specific receptors for the imported proteins (13, 14, 15, 16, 17) . Binding of precursors to these receptors is one of the earliest steps of protein import into the mitochondria. Does the import of MTF1 involve any proteinaceous receptor on the outer surface of the mitochondria? Mitochondria were pretreated with trypsin and then used for the assay of import of MTF1. F and ATP/ADP carrier proteins were used as controls. The results depicted in Fig. 2 indicate that when untreated mitochondria were used for the import assay, the F produced a mature form (smaller in size than the precursor) (lane1), which is resistant to exogenous proteinase K (lane2), indicating its import into mitochondria. The ADP/ATP carrier, on the other hand, is not reduced in size upon incubation with mitochondria (lane1) under import assay conditions, since it does not have a cleavable presequence, but it is translocated to a site within the mitochondria that renders it resistant to proteinase K (lane2). When trypsin- (30 µg) treated mitochondria were used, both F and ADP/ATP carrier failed to be imported into the mitochondria as evidenced by the absence of protease protected bands (lanes3 and 4). MTF1, on the other hand, was able to be transported into both untreated (lanes1 and 2) and trypsin-pretreated mitochondria (lanes3 and 4). MTF1 was found to be imported also into the mitochondria pretreated with 200 µg of trypsin (data not shown). This observation suggests that its import probably does not require a receptor on the outer surface of the mitochondria.


Figure 2: Import of MTF1 is not sensitive to Trypsin pretreatment. 40 µl of lysate containing the labeled protein (either F or AAC, or MTF1) were incubated with 150 µg of mitochondrial protein either without pretreatment (lanes1 and 2) or pretreated with 30 µg of trypsin (as described under ``Experimental Procedures'') (lanes3 and 4) in a 200 µl of import reaction buffer at 25 °C for 30 min. After import, the reaction mix was divided into two equal parts. 40 µg of proteinase K was added to one part (lanes2 and 4), incubated on ice for 30 min, followed by the addition of 1 mM PMSF. Mitochondria were reisolated in each case and analyzed by SDS-PAGE followed by fluorography.



Since the MTF1 is resistant to proteinase K after import in trypsin-pretreated mitochondria, we examined the sensitivity of in vitro synthesized labeled MTF1 to proteases by incubating with different concentrations (5-40 µg) of proteases viz. proteinase K and trypsin. Newly synthesized MTF1 was found to be sensitive to even 5 µg of proteinase K, although more than 5 µg of trypsin was needed to degrade it (data not presented). This indicates that the results presented in Fig. 1 and Fig. 2are not simply attributable to the ability of MTF1 in the absence of detergent to assume a proteinase-resistant conformation.

MTF1 Import Is Not Dependent on a Membrane Potential

Translocation of precursor proteins into and across the inner membrane depends on energization of the membrane provided by the electrochemical potential. To determine if MTF1 requires a membrane potential for its import, CCCP, an uncoupler of oxidative phosphorylation, and valinomycin, an ionophore, were used during the import. As a control for the effectiveness of these two agents, the import of F subunit was studied alongside that of MTF1. The results are depicted in Fig. 3. In the absence of either agent, mitochondria produces mature F (lane1), which is protease-protected (lane2). But in the presence of both CCCP and valinomycin, mature form is not produced; rather, there is accumulation of the precursor (lanes3 and 5), which is sensitive to proteinase K (lanes4 and 6), indicating that the import of F is dependent on the membrane potential. However, MTF1 is transported into a protease-protected location within the mitochondria in the presence of both CCCP (lanes3 and 4) and valinomycin (lanes5 and 6), suggesting that MTF1 import is independent of the membrane potential. The effect of inhibition of the matrix-localized protease, responsible for the cleavage of the presequences from the imported mitochondrial proteins, was examined using 1,10-phenanthroline and EDTA. Under these conditions, F failed to be imported into the mitochondria, while the import of MTF1 was not at all affected (lanes7 and 8).


Figure 3: MTF1 can be imported in the absence of membrane potential. 40 µl of lysate containing labeled protein (either F or MTF1) was incubated with 150 µg of mitochondrial protein in a 200 µl of import reaction buffer in the absence of any agent (lanes1 and 2) or in the presence of either 40 µM CCCP (lanes3 and 4), or 5 µM valinomycin (lanes5 and 6), or 250 µM 1,10-phenanthroline and 5 mM EDTA (lanes7 and 8) at 25 °C for 30 min. After import, the reaction mixture was divided into two equal parts, and 40 µg of proteinase K was added to one of them (lanes2, 4, 6, and 8) and incubated on ice for 30 min followed by the addition of 1 mM PMSF. The mitochondria were reisolated in each case and analyzed by SDS-PAGE followed by fluorography.



Import of MTF1 Does Not Need ATP

It has been shown that protein import into the mitochondria requires ATP along with an energized inner membrane (18) . Pfanner et al.(19) suggested that ATP is necessary both in the cytosol and in the matrix (a) on the cytosolic side for the maintenance of the translocation competent conformation of the precursor and (b) for refolding and sorting of the precursors that involve chaperone-like components. To investigate the requirement of ATP for the import of MTF1, we depleted the mitochondria and the rabbit reticulocyte lysate (containing the labeled precursor) of ATP by treating them with apyrase. The effectiveness of functional ATP depletion was assessed by examining the import of F in parallel with that of MTF1. When untreated mitochondria were used in the presence of exogenous ATP and NADH, F was found to be cleaved, and the mature form is accumulated inside the mitochondria (Fig. 4, lanes2 and 3). This was observed even in the absence of exogenous ATP and NADH (lanes4 and 5) since the mitochondria and the rabbit reticulocyte lysate contain endogenous ATP. When this ATP was removed by apyrase treatment, F failed to be imported (lanes6 and 7). MTF1, on the other hand, was imported even in apyrase-treated mitochondria (lanes6 and 7), indicating that its import requires little if any ATP.


Figure 4: MTF1 can be imported in the absence of ATP. 40 µl of lysate containing the labeled protein (either F or MTF1) was incubated with 150 µg of mitochondrial protein in a 200-µl import buffer in the presence (lanes2 and 3) or absence (lanes4-7) of 2 mM ATP and 1 mM NADH and incubated at 25 °C for 30 min. In the case of lanes6 and 7, both the mitochondria and the lysate, containing the labeled protein, were pretreated with apyrase as described under ``Experimental Procedures.'' After the reaction, each reaction mix was divided into two equal parts, and one of them was treated with 40 µg of proteinase K (lanes3, 5, and 7) incubated on ice for 30 min followed by the addition of 1 mM PMSF. The mitochondria were reisolated and analyzed by SDS-PAGE followed by fluorography.



Import of MTF1 Occurs Even at Low Temperature

Temperatures lower than 20 °C reduce the transfer of precursor proteins across the two mitochondrial membranes. Translocation intermediates are trapped by performing import at lower temperatures (20, 21) . MTF1 import was examined at different temperatures viz. 2, 10, and 25 °C. The extent of import was found to be the same in each case (Fig. 5), indicating that the import of MTF1 is not reduced by carrying out the import at low temperature.


Figure 5: Low temperature does not have any effect on the import of MTF1. 40 µl of lysate containing the labeled MTF1 was incubated with 150 µg of mitochondrial protein in a 200-µl import buffer at either 2 °C (lanes2 and 3), 10 °C (lanes4 and 5), or 25 °C (lanes6 and 7) for 30 min. Each reaction mixture was then divided into two equal parts, and one of them (lanes3, 5, and 7) was incubated with 40 µg of proteinase K on ice for 30 min followed by the addition of 1 mM PMSF. The mitochondria were reisolated and analyzed by SDS-PAGE followed by fluorography.



The Amino-terminal Two-thirds of MTF1 Contains the Mitochondrial Targeting Sequence

Though many mitochondrial proteins are synthesized with amino-terminal presequences that are removed proteolytically within the mitochondrial matrix, some of these proteins are synthesized without cleavable presequences, and they include their targeting signals within their mature peptide (6) . Since MTF1 is translocated into the mitochondria without reduction in size, we began to assess whether the targeting signal was present at the amino or carboxyl-terminal half of the MTF1 gene product. The MTF1 gene was truncated from its 3`-end with unique restriction endonucleases, and the resulting 5`-fragments were used for the coupled transcription, translation, and import assay. Digestion of the MTF1 gene (1 kilobase) with BglII and BamHI produces 5` 889- and 671-base pair fragments, respectively. The corresponding protein of the longer fragment (BglII) (obtained by coupled transcription and translation using the fragment as a template), was imported into the mitochondria as evidenced by its protection from proteinase K digestion (Fig. 6, lanes2 and 3). In the case of the BamHI fragment, only a very small proportion of the available protein was protected from protease digestion (lane3), suggesting very limited import. Both products were fully sensitive to proteinase K when the mitochondria were reisolated after import and lysed with Nonidet P-40 before the addition of proteinase K, indicating that these peptides had probably been transported to the interior of the mitochondria (lane4). It is notable, however, that the efficiency of import with the truncated protein derived from the BglII fragment was not as great, as is the case with the intact protein.


Figure 6: Amino-terminal fragments of MTF1 can be imported into isolated mitochondria. The MTF1 gene was digested with either BglII or BamHI, and the resulting fragments were used for in vitro transcription and translation as described under ``Experimental Procedures.'' 60 µl of the lysate containing the labeled protein was incubated with 225 µg of mitochondrial protein in a 300-µl import buffer at 25 °C for 30 min. After import, the import reaction mixture was divided into three parts (lanes2, 3, and 4). One of them was treated with 40 µg of proteinase K and incubated on ice for 30 min (lane3) followed by the addition of 1 mM PMSF. Mitochondria were reisolated from another part (lane4), lysed with 1% Nonidet P-40 and 80 mM KCl, and then treated with 40 µg of proteinase K. Mitochondria were reisolated from the other two, and all of the samples were analyzed by SDS-PAGE followed by fluorography.



Newly Imported MTF1 Is Internal to the Inner Mitochondrial Membrane

Since MTF1 can be imported into mitochondria devoid of ATP, in the absence of membrane potential and even at low temperature, the question arises, in which of the four mitochondrial compartments (outer membrane, inner membrane, intermembrane space, and matrix) was MTF1 located after import under those conditions? Mitochondria, after import of MTF1, were treated with two different concentrations of digitonin followed by treatment with proteinase K. AAC protein, which resides on the inner membrane facing the inter membrane space, was used to validate the fractionation. Fig. 7A shows that AAC, after import at 25 °C in the presence of a membrane potential and ATP (lane1), was protected from proteinase K (lane2) but was degraded by proteinase K when the mitochondria were treated with either 0.25% digitonin (lane4) or 0.5% digitonin (lane6), showing that the AAC is accesssible to proteinase K after digitonin treatment. Under the same conditions of import (Fig. 7B), MTF1 was protected from proteolysis even after treatment with 0.5% digitonin. In view of the unusual features of MTF1 import, we checked that the protein was similarly resistant to proteolysis even in the presence of valinomycin (Fig. 7C) or CCCP (Fig. 7D) or in the absence of ATP (Fig. 7E) and after treatment with 0.5% digitonin (lane6). When the import was carried out at low temp (5 °C) (Fig. 7F), in the presence of CCCP (Fig. 7G) or valinomycin (Fig. 7H), in the absence of ATP (Fig. 7I), or in the absence of ATP but in the presence of CCCP (Fig. 7J), MTF1 was still found to be resistant to proteinase K after treatment with both 0.25 and 0.5% digitonin. These data indicate that MTF1 was imported across the inner membrane even when there was little or no electrochemical gradient, no ATP, and at low temperature.


Figure 7: Use of digitonin to subfractionate mitochondria after import. 120 µl of lysate containing labeled protein (either MTF1 or AAC) was incubated with 450 µg of mitochondrial protein in a 600-µl import buffer at 25 °C (A-E) or at 5 °C (F-J) for 30 min. A, B, and F, in the presence of exogenous ATP and NADH; C and H, in the presence of exogenous ATP, NADH, and 5 µM valinomycin; D and G, in the presence of exogenous ATP, NADH, and 40 µM CCCP; E and I, in the absence of exogenous ATP and NADH (the mitochondria and the lysate were treated with apyrase before import, as described under ``Experimental Procedures,'' to remove the endogenous ATP); J, in the absence of exogenous ATP and NADH but in the presence of 40 µM CCCP (the mitochondria and the lysate were treated with apyrase before import to remove the endogenous ATP). After import, the reaction mix was divided into six equal parts (lanes1-6). Two parts (lanes3 and 4) were treated with 0.25% digitonin, while two others (lanes5 and 6) were treated with 0.5% digitonin as described under ``Experimental Procedures.'' 40 µg of proteinase K was added to samples displayed in lanes2, 4, and 6 (after digitonin treatment) and incubated on ice for 15 min followed by the addition of 1 mM PMSF. The mitochondria and the mitoplasts were reisolated and analyzed by SDS-PAGE followed by fluorography.



Newly Imported MTF1 Is Present in the Mitochondrial Matrix

Next we have fractionated the mitochondria containing the newly imported MTF1 to ascertain whether it is attached to the inside of the inner membrane or present in the matrix. The results given in Fig. 8 show that imported MTF1 is mostly present in the soluble supernatant indicating its matrix localization. This corresponds to the distribution of citrate synthase, an authentic matrix component (data not shown).


Figure 8: Localization of imported, proteinase K resistant MTF1. Lane1, 10 µl of reticulocyte lysate containing the labeled MTF1; lanes2-5, 700 µg of mitochondrial protein were incubated with 140 µl reticulocyte lysate containing labeled MTF1 in a 700-µl import reaction buffer containing ATP and NADH at 25 °C for 30 min. After import, a 100-µl aliquot was removed (lane2). 240 µg of Proteinase K was added to the rest, which was incubated on ice for 30 min followed by the addition of 1 mM PMSF. A 100-µl aliquot was removed (lane3). The mitochondria from the remaining 500-µl reaction mix were reisolated and mixed with 4.5 mg of untreated mitochondrial protein, which was then subjected to submitochondrial fractionation according to the method of Daum et al. (7) as follows. The mitochondria were suspended in a buffer containing 0.1 M sorbitol, 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1 mM PMSF and incubated on ice for 20 min followed by sonication for 15 s at 50 watts. The sonication was repeated 3 times with 30 s of cooling between the sonications. The resulting extract was centrifuged at 35,000 rpm for 1 h in Ti-50 rotor at 4 °C to separate soluble supernatant from the membrane fraction. The proteins from the soluble supernatant were precipitated by ice-cold trichloroacetic acid. Equivalent amounts of proteins of membrane fraction (lane4) and soluble supernatant (lane5) were used. The mitochondria were reisolated from the samples displayed in lanes2 and 3, and all of the samples were analyzed by SDS-PAGE followed by fluorography.



MTF1 Can Be Imported into Mitoplasts

It has recently been suggested that the outer and inner membranes of mitochondria contain independent import machineries (22, 23) . So we examined whether mitoplasts, which lack their mitochondrial outer membranes and hence their outer membrane receptors, are competent to import MTF1. The results given in Fig. 9A show that AAC can be imported successfully into mitochondria (lane3) but not into the mitoplasts (lane5). MTF1, on the other hand, is imported as efficiently into the mitoplasts (lane5) as it is into the mitochondria (lane3). The results illustrated in Fig. 9B show that MTF1 can be imported into the mitoplasts even at low temperature. The results of the experiments with mitoplasts indicate that MTF1 can be transported across the inner membrane of the mitochondria without the need for an intact outer membrane.


Figure 9: Import of MTF1 into the mitoplasts. A, 40 µl of lysate containing labeled protein (either MTF1 or AAC) were incubated with 150 µg of either mitochondria (lanes2 and 3) or mitoplasts (lanes4 and 5) in a 200-µl import buffer at 25 °C for 30 min. Each import reaction mixture was then divided into two parts, and one of them (lanes3 and 5) was treated with 40 µg of proteinase K on ice for 30 min followed by the addition of 1 mM PMSF. The mitochondria or the mitoplasts were reisolated and analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography. B, 40 µl of lysate containing the labeled MTF1 were incubated with 150 µg of mitoplasts in 200 µl of import buffer at different temperatures. The import reaction mixture was then divided into two equal parts, one of which (lanes3, 5, and 7) was treated with 40 µg of proteinase K on ice for 30 min followed by the addition of 1 mM PMSF. The mitoplasts were reisolated and analyzed by SDS-PAGE followed by fluorography. C, 80 µl of lysate containing the labeled MTF1 were incubated with 300 µg of either mitochondria (mt) (lanes2-5) or mitoplasts (mp) (lanes6-13) in 400 µl of import buffer either at 25 or 5 °C for 30 min. The import reaction mix was then divided into four equal parts and treated with either proteinase K (lanes3, 4, 7, 8, 11, and 12) on ice for 30 min followed by the addition of PMSF (1 mM final concentration) or trypsin (lanes5, 9, and 13) at 4 °C followed by the addition of trypsin inhibitor (10 the concentration of trypsin). Mitochondria or mitoplasts were reisolated and analyzed by SDS-PAGE followed by fluorography.



A careful examination of most of the figures so far presented indicates that proteinase K treatment modestly reduces the length of MTF1 after import into either mitochondria or mitoplasts. Similar reduction was observed when 5 µg of proteinase K instead of 40 µg was used. However, no reduction was observed when 40 µg of trypsin was used instead of proteinase K (Fig. 9C). Reduction was also not observed when truncated MTF1 (either BglII or BamHI fragments) were used for import into mitochondria (Fig. 6) or mitoplasts (data not shown). When mitoplasts, prepared from steady-state mitochondria, were treated with proteinase K followed by blotting and probing with antiMTF1 antibody, we did not see any reduction in size of the endogenous MTF1 (data not presented).

Sorting of MTF1 Is Independent of hsp60

There are two types of sorting pathways for the proteins that are imported into the mitochondria. The conservative sorting pathway involves ATP-dependent interaction with the hsp60. The proteins that follow this pathway have amino-terminal cleavable presequences and interact with the MOM19 receptor preliminary to their entry into the mitochondria. The nonconservative sorting pathway does not involve the interaction with the hsp60. ADP/ATP carrier, which does not have a cleavable presequence, follows this pathway for its import from outer to the inner membrane via MOM72 (13, 14, 24) . The role of hsp60 in the import of MTF1 was examined taking advantage of a temperature-sensitive mutation in the hsp60 gene (mna2-1) (25) . In separate studies, we have shown that this mutant is defective in the import of several proteins e.g. F subunit of ATP synthase, that are sorted by an hsp60-dependent pathway.() MTF1 was imported at 37 °C into the mitochondria isolated from the mna2-1 pet mutant grown at 37 °C (the nonpermissive temperature). The results depicted in Fig. 10show that the import of MTF1 in contrast to that of F subunit (data not shown) is intact in mna2-1 mitochondria at the restrictive temperature, indicating an hsp60-independent pathway for MTF1 import into the mitochondria.


Figure 10: Import of MTF1 into the mitochondria isolated from the mna2-1 mutant grown at restrictive temperature. Identical amounts of labeled MTF1 protein were used to import (in a 100-µl import reaction buffer) into the mitochondria (75 µg) isolated from the wild-type yeast 124 (lanes2-5) and the mutant mna2-1 (lanes6-9) grown at 37 °C. The import was carried out at either 25 °C (lanes2, 3, 8, and 9) or at 37 °C (lanes4-7) for 30 min. Two identical sets were used for import. One of them after import was treated with 40 µg of proteinase K (lanes3, 5, 7, and 9) on ice for 30 min. The mitochondria were reisolated and analyzed by SDS-PAGE followed by fluorography.



DISCUSSION

We have studied the requirements for the mitochondrial import of the mitochondrial transcription factor. Our results indicate that there are no specific requirements for this import. There is no evidence for the requirement of an outer membrane receptor, for an electrochemical gradient, for ATP, for hsp60. MTF1 is efficiently imported into trypsin-treated mitochondria and into mitoplasts lacking the bulk of the outer membrane. The independence of MTF1 import of each of the factors normally involved in the import of prototypic proteins destined for mitochondria suggests that this protein may exist in an extended or loosely folded conformation both in its transport competent form and perhaps also in its functional form.

While we have not pinpointed the sequence(s) responsible for mitochondrial localization, we have evidence that it is present at the amino-terminal half of the protein. We are currently engaged in studying the more precise definition of the targeting sequence.

There are other proteins that enter the mitochondria independent of one or more features that characterize the usual protein import mechanism or pathway. But none of them is as fully independent of the features upon which mitochondrial import depends, as is the case for MTF1. Import of cytochrome c into the inter membrane space is independent of a surface receptor (26, 27) . Import of subunit Va of cytochrome c oxidase into the inner membrane also is independent of a proteinaceous surface receptor (11) . Transport of apocytochrome c across the outer membrane is not dependent on the membrane potential but requires functional cytochrome c heme lyase. Import of subunit Va, on the other hand, is dependent on the membrane potential for its import, although its requirement for ATP is very low and it is completely imported at low temperature. Our results, taken together, strongly indicate that the import pathway of MTF1 is quite unusual compared with that of the majority of proteins studied so far and is distinct even from the pathways used by either cytochrome c or subunit Va of cytochrome c oxidase, although it does share some but not all of the import features with each of the latter proteins.

Apocytochrome c, requiring no mitochondrial surface receptor, probably inserts spontaneously into the mitochondrial lipids of the outer membrane, dependent mainly on the anionic lipids (27). It crosses the mitochondrial membrane reversibly and is fixed inside the mitochondria by reaction with heme catalyzed by cytochrome c heme lyase. It remains to be established whether similar features characterize the transport of the transcription factor. If it associates spontaneously with mitochondrial lipid, it does not appear to distinguish between the lipids of the outer membrane and inner mitochondrial membranes, since the protein is imported by mitoplasts lacking intact outer membrane. This experiment would not, however, distinguish transport through the outer membrane-inner membrane contact sites, which are probably preserved in the mitoplasts. It remains to be established whether MTF1 gene product crosses mitochondrial membranes specifically or whether it can readily cross other cellular or organellar membranes. Further experiments along these lines are in process. If the mitochondrial transcription factor is able to cross other membranes easily, it would be necessary to postulate some fixation event to account for its mitochondrial targeting as is the case for cytochrome c. Once imported, the transcription factor may be fixed in the mitochondrial matrix by one of three possible events: interaction with another macromolecule in the mitochondrial matrix (DNA or protein), post-translational chemical modification (e.g. acylation), or postimport folding catalyzed by an intramitochondrial chaperone. Each of these possibilities is being explored in further experiments.

The findings on MTF1 import are thus quite surprising. Since much of the electrochemical gradient and ATP are required for the preservation of a translocation-competent conformation and since neither of these conditions is required for MTF1 import, one might postulate that MTF1 naturally assumes a translocation-competent conformation. This could only be established by physical analysis of the MTF1 synthetic product or by strategic mutagenesis.

Another feature that deserves comment and may relate to the conformation for translocation is the observation that proteinase K but not trypsin digestion reveals a product that is slightly, but reproducibly shortened. There is good reason to believe that this does not represent cleavage of the amino-terminal pretargeting or targeting sequence. First, the product isolated from intact mitochondria, when compared with the sequence specified by the cloned gene, lacks only the amino-terminal methionine. Second if removal of the presequence was the basis for the small reduction in size, this should be revealed by trypsin, as well as proteinase K proteolysis. Third, inhibition of the matrix metalloprotease thought to be involved in processing of recently imported mitochondrial precursors should prevent the observed reduction in protein size. This was not the case. Since the newly imported protein is in the matrix and not attached to the inner membrane, even transiently, it seems that this reduction in size cannot be explained as a transport intermediate that assumes a hairpin configuration with a small portion of the carboxyl terminus accessible to proteinase K. The basis for this apparent shortening must await further experimentation.


FOOTNOTES

*
This work was supported by Grant HL04442 from the United States Public Health Service, National Heart, Lung, and Blood Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: CCCP, carbonyl cyanide m-chlorophenylhydrazone; AAC, ADP/ATP carrier; MOPS, 4-morpholinepropanesulfonic acid; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis.

A. Sanyal, B. Tung, and G. S. Getz, manuscript in preparation.

A. Sanyal and G. S. Getz, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Charlene Murphy for help in the preparation of the manuscript.


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