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Import of Mitochondrial Carriers Mediated by Essential Proteins of the Intermembrane Space

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Science  16 Jan 1998:
Vol. 279, Issue 5349, pp. 369-373
DOI: 10.1126/science.279.5349.369

Abstract

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

Most proteins imported to mitochondria are synthesized with a cleavable NH2-terminal targeting sequence and are sorted to their correct intramitochondrial location by the dynamic interaction of distinct transport systems in the outer and inner membranes (1). The TIM system in the inner membrane consists of two integral membrane proteins, Tim17 and Tim23, which make up the inner membrane import channel. Complete translocation into the matrix is coupled to adenosine triphosphate (ATP) hydrolysis and is mediated by Tim44, mHsp70, and GrpE. However, some of the most abundant inner membrane proteins, such as the metabolite carriers, are synthesized without a cleavable NH2-terminal presequence and therefore do not engage with the Tim23 channel. It has been suggested that import of these proteins is directed by one or more internal targeting signals (2), but the exact mechanism is still poorly defined. In the cytosol of the yeast Saccharomyces cerevisiae, chaperones escort these insoluble carrier proteins preferentially to the outer membrane receptors Tom37 and Tom70 (3). The carriers then move through the TOM channel in the outer membrane and insert into the inner membrane, bypassing the ATP-dependent Tim23 system, which transports proteins across the inner membrane (4, 5).

Here we report that the transfer of insoluble carrier proteins from the TOM system of the outer membrane across the aqueous intermembrane space to the inner membrane is mediated by Tim10p and Tim12p, two cysteine-rich proteins of the intermembrane space. The two proteins are 35% identical and are essential for viability. They were discovered as multicopy suppressors of mitochondrial RNA splicing defects and were initially termed Mrs11p and Mrs5p (6-8).

To identify the function of Tim10p and Tim12p, we constructed temperature-sensitive mutants by in vitro mutagenesis (9). Haploid yeast cells expressing a temperature-sensitivetim10-1 or tim12-1 protein grew at wild-type rates at 25°C but stopped growing 6 to 8 hours after a shift to 37°C on fermentable as well as nonfermentable carbon sources. Return to 25°C did not restore growth (10). Mitochondria purified from heat-arrested tim10-1 cells contained drastically reduced steady-state concentrations of several carriers located in the inner membrane—including the ADP-ATP carrier (AAC), the phosphate carrier (PiC), and the dicarboxylate carrier (DiC) (11)—as well as of Tim22p (12) (Fig.1A). A similar but milder defect was found in mitochondria from the heat-arrested tim12-1 mutant, with the exception of Tim22p, which was not detectable. Both arrested mutants had wild-type levels of proteins sorted to the outer membrane, matrix, intermembrane space, and inner membrane that were unrelated to the carrier family. Thus, the defect in both mutants was specific for the metabolite carrier family and for Tim22p.

Figure 1

Tim10p and Tim12p are required for import of mitochondrial carrier proteins and Tim22p. (A) The parental wild-type strain (WT) and the two mutant strains were grown in lactate medium for 16 hours at 25°C and then shifted for 8 hours to 37°C. Mitochondria were isolated and analyzed by SDS-PAGE and immunoblotting with monospecific rabbit antisera for AAC, porin, cytochrome b2, and Tim11p (20, 40, and 80 μg loaded) and PiC, DiC, and Tim22p or Tim23p (50, 100, and 200 μg loaded). Blots were decorated with [125I]–protein A. (B) Precursors were synthesized in vitro and incubated at 25°C with fully energized wild-type or mutant mitochondria, followed by protease K treatment, SDS-PAGE, and fluorography (13). Aliquots were analyzed for import after 2.5, 5, 10, and 15 min; p, precursor; i, intermediate form; m, mature form; −ΔΨ, absence of a membrane potential; import for 15 min into wild-type mitochondria uncoupled by 25 μM FCCP.

To determine whether the import of carriers was impaired, we tested mitochondria from the heat-arrested mutants for their ability to import radiolabeled precursors synthesized in vitro (13) (Fig. 1B). Import was assessed by protection from added protease, which removes nonimported precursor. The observed import defects mirrored the deficiency in steady-state levels: import of AAC and PiC was inhibited by 85% in tim10-1 mitochondria and by 50% intim12-1 mitochondria, whereas import of Tim22p was inhibited by 85% in both tim10-1 and tim12-1 mitochondria. All of the proteins whose import into isolated mitochondria was affected by the two mutations lack a cleavable targeting signal. In contrast, import or proteolytic processing of mitochondrial precursor proteins with a cleavable NH2-terminal targeting presequence was unaffected by either mutation, regardless of whether the precursors were targeted to the intermembrane space or to the matrix.

Import of the AAC precursor into wild-type mitochondria occurs in several discrete stages (5). In stage 1, the soluble carrier, escorted by cytosolic chaperones, binds to the mitochondrial import receptors. In stage 2, it partially (and probably reversibly) inserts across the TOM pore in the outer membrane, becoming partly inaccessible to proteinase K. In stage 3, the carrier accumulates on the outer surface of the inner membrane, becoming inaccessible to proteinase K in intact mitochondria, but not in mitoplasts (mitochondria whose outer membrane has been selectively ruptured). In stage 4 (which requires an electric potential across the inner membrane), the carrier inserts fully into the inner membrane and is then unable to be extracted with alkali, with only a small NH2- or COOH-terminal part accessible to protease in mitoplasts. In stage 5, dimerization of the inserted carrier in the inner membrane completes the process.

To determine the specific stage at which the import of carriers was blocked in the tim10-1 and tim12-1 mutants, we allowed mitochondria from the heat-arrested cells to import AAC and then either alkali-extracted or converted them to mitoplasts in the presence of protease. In agreement with published results, AAC inserted into the inner membrane of wild-type mitochondria only in the presence of an inner membrane potential, the inserted protein was resistant to protease in mitochondria and mitoplasts, and the fully inserted carrier was not extracted by alkali (Fig. 2, A and B). In contrast, import of the carrier into mitochondria from heat-arrested tim12-1 cells was blocked at stage 3 even in the presence of an inner membrane potential: the imported carrier was resistant to protease in intact mitochondria (Fig. 2A) but not in mitoplasts (Fig. 2B) and remained largely alkali-extractable (Fig. 2A). (Nonspecific losses of alkali-extractable AAC most likely were caused by nonspecific adherence to the tube wall.) Import of multispanning carrier proteins into fully energized tim12-1 mitochondria thus resembled the incomplete import into wild-type mitochondria lacking an inner membrane potential.

Figure 2

Tim12p is required for insertion of AAC into the inner membrane, whereas Tim10p is required for translocation of the AAC across the outer membrane. (A) AAC was synthesized in vitro and incubated with wild-type (WT) and tim12-1 mitochondria for 8 min at 25°C in the presence or absence of a membrane potential (ΔΨ). Mitochondria were then divided into equal aliquots that were either left untreated (−) or treated with proteinase K (PK, 30 or 200 μg/ml) for 15 min at 4°C, followed by 1 mM PMSF. Where indicated, mitochondria were extracted with 100 mM Na2CO3 for 30 min at 4°C. After centrifugation at 100,000g for 15 min, pellet (P) and supernatant (S) were precipitated with 10% trichloroacetic acid (TCA) and analyzed by SDS-PAGE and fluorography. Standard (STD): 10% of the radioactive precursor present in each assay. (B) Import was as in (A). The designated samples were then treated with trypsin (100 μg/ml) for 30 min at 4°C, followed by addition of trypsin inhibitor (200 μg/ml). Two aliquots were converted to mitoplasts (MP) in the absence or presence of proteinase K (50 μg/ml), separated into pellet (P) and supernatant (S), and then analyzed as in (A). (C) Import into tim10-1mitochondria was as in (A). After import, three equal aliquots were taken: one was left untreated (−), whereas the other two were treated with proteinase K (30 or 200 μg/ml) for 15 min at 4°C, followed by addition of 1 mM PMSF. All samples were then analyzed as in (A).

Import of AAC into mitochondria from the heat-arrestedtim10-1 mutant was blocked at an earlier stage than import into tim12-1 mitochondria: the precursor was bound to the mitochondria, but most of it remained protease-accessible (Fig. 2C), and what little was imported remained alkali-extractable (11). The import block caused by loss of Tim10p function thus occurs before or at stage 2, suggesting that Tim10p is required for transfer of the carrier across the outer membrane.

To show directly that Tim10p interacted with AAC during import, we imported radiolabeled AAC into fully energized or uncoupled mitochondria, removed nonimported protein by protease treatment, reacted the mitochondria with a cleavable cross-linker, and analyzed the radioactive cross-linked products by SDS–polyacrylamide gel electrophoresis (PAGE) and fluorography (14). Import into fully energized wild-type mitochondria yielded only a low percentage of cross-linked products, presumably because the imported carrier was rapidly inserted into the inner membrane (Fig.3A). Little cross-linking was detected with mitochondria from the tim10-1 mutant (Fig. 3A), because the carrier was not transported across the outer membrane. In contrast, at least two cross-linked products were found after import into fully energized or uncoupled tim12-1 mitochondria or into uncoupled wild-type mitochondria. Each of the two cross-linked products was as abundant as the non–cross-linked AAC, and their apparent masses were 10 and 20 kD greater than that of AAC. Both products could be immunoprecipitated by monospecific antiserum against Tim10p in uncoupled wild-type mitochondria (Fig. 3B) and tim12-1mitochondria (11), indicating that they contained Tim10p. Upon cleavage with 2-mercaptoethanol, the cross-linked species yielded only the radioactive 34-kD band of AAC (11). Thus, loss of Tim12p function, or uncoupling of wild-type mitochondria, caused accumulation of Tim10p-bound AAC inside the outer membrane. The large cross-linked bands may contain Tim12p, but antibodies against Tim12p could not establish this point with certainty.

Figure 3

Tim10p can be cross-linked to the AAC precursor during import and forms a complex with Tim12p and Tim22p. (A) Import was performed with wild-type,tim12-1, and tim10-1 mitochondria in the presence or absence of a membrane potential (ΔΨ), and mitochondria were re-isolated and divided as follows. One-quarter was precipitated with TCA (−DSP); the rest (1 mg/ml) was cross-linked with DSP (14). Asterisks mark the location of two major cross-linked products of about 44 and 54 kD. (B) AAC was imported into uncoupled wild-type mitochondria. After import, one-seventh of the assay was precipitated with TCA (−DSP), and the remainder was incubated with DSP. Two-sevenths were precipitated with TCA after cross-linking, and the remainder was denatured with SDS and subjected to immunoprecipitation either with preimmune antiserum (CS) or with antiserum to Tim10p (14). “X” marks the two major cross-linked products that were detected in (A), and the asterisk marks another immunoprecipitated fragment that may contain Tim10p. (C) Mitochondria from a strain expressing a COOH-terminal hexahistidine-tagged Tim10p [Tim10H6p (14)] were solubilized at 2 mg/ml by 0.5% digitonin. As a control, 150 μg of extract was withdrawn (T), and 1 mg was incubated with Ni2+-agarose beads. The beads were washed, and bound proteins were eluted with 1% SDS (B). To assess the effectiveness of binding, 150 μg of the unbound proteins (S) was also analyzed. Proteins co-isolating with the hexahistidine-tagged Tim10p were identified and quantified by immunoblotting. The fraction of bound and unbound protein is given below each panel. Mitochondria from the parental wild-type strain were analyzed as a negative control.

To identify proteins that interact with Tim10p and Tim12p, we searched for yeast genes whose overexpression abrogated the conditional lethality of the tim10-1 and tim12-1 mutations (15). No suppressor was identified for thetim10-1 mutation, but we isolated one strong suppressor for the tim12-1 mutation. After subcloning, this suppressor proved to be the TIM22 gene. Overexpression ofTIM22 allowed the tim12-1 mutant to grow at the restrictive temperature 37°C but was without effect on the conditional lethality of the tim10-1 mutant or atim12 null strain (11). None of the genes for other known components of the mitochondrial import system, includingTIM23 or TIM17, were identified in this multicopy suppressor screen. Even though Tim10p and Tim12p are 35% identical, overexpression of one protein did not suppress the lethality caused by the temperature sensitivity of the other (11).

The results presented so far suggested that Tim10p, Tim12p, and Tim22p interact functionally and genetically. To test for a physical interaction between Tim10p, Tim12p, and Tim22p, we constructed a yeast strain in which all Tim10p molecules had a COOH-terminal hexahistidine tag (16). This strain grew as well as the parental wild type under all conditions tested, suggesting that the tag did not abolish the function of Tim10p in vivo. Essentially all of the tagged Tim10p present in digitonin-solubilized mitochondria was recovered on the Ni2+-agarose beads (Fig. 3C). More than 90% of the Tim12p and 80% of the Tim22p present in the mitochondrial lysate co-purified with the tagged Tim10p. No such co-purification was detected if Tim10p was untagged, and inner membrane proteins such as AAC, Tim23p, Tim11p, Tim44p, and Afg3p (11, 17) did not co-purify with the tagged Tim10p (Fig. 3C). Thus, the genetic and biochemical data indicated that Tim10p, Tim12p, and Tim22p are three components of a multisubunit complex that mediates import and insertion of multispanning carrier proteins into the inner membrane.

Tim10p and Tim12p perform distinct functions in the import of mitochondrial carriers because overexpression of Tim10p did not substitute for loss of Tim12p and vice versa. Tim10p acted before Tim12p: Loss of Tim10p function blocked transport across the outer membrane, whereas loss of Tim12p function blocked insertion into the inner membrane. Tim10p was only loosely associated with the complex because most of it was solubilized when mitochondria were converted to mitoplasts (7). Association of Tim10p with the inner membrane may be reversible, allowing Tim10p to (i) bind precursor whose COOH-terminus is still in the outer membrane channel or exposed on the outer surface and (ii) transfer the bound precursor to Tim12p. We suggest the following model for the import pathway of a multispanning carrier from the mitochondrial surface into the inner membrane (Fig.4). The carrier, after binding to mitochondrial import receptors, passes through the outer membrane pore and binds to Tim10p; this step may be reversible. The carrier is then transferred to Tim12p, which is associated with Tim22p on the outer face of the inner membrane. The membrane-embedded Tim22p finally mediates the correct insertion of the carrier into the inner membrane.

Figure 4

Model for the import of mitochondrial carrier proteins. Proteins 5, 6, 7, 20, 22, 37, 40, and 70 are members of the TOM complex. Proteins 11, 17, 23, and 44 are members of (or closely adjacent to) the Tim23p-Tim17p complex that mediates import of precursors carrying a cleavable NH2-terminal matrix-targeting sequence. Proteins 10, 12, and 22 are members of the Tim22 complex that mediates import of multispanning carrier proteins into the inner membrane. A carrier precursor exiting the TOM channel is captured by Tim10p in the intermembrane space and delivered to Tim12p, which is bound to Tim22p at the outer face of the inner membrane. Transfer to Tim12p triggers the Tim22p-dependent insertion of the multispanning carrier into the inner membrane.

Because Tim10p and Tim12p are required for insertion of Tim22p, which in turn is required for the insertion of carrier proteins, Tim10p and Tim12p might in principle only be needed to maintain wild-type Tim22p levels. This model cannot be excluded, but it cannot readily account for our findings that partly imported AAC can be cross-linked to Tim10p in uncoupled wild-type mitochondria and that overexpression of Tim22p fails to suppress loss of Tim10p function. Thus, we favor the view that Tim10p and Tim12p interact directly with each of the different carrier precursors and that Tim22p, because it is a multispanning inner membrane protein, uses this same import pathway.

Proteins similar to Tim10p and Tim12p are present inSchizosaccharomyces pombe and Caenorhabditis elegans (10), suggesting that this second TIM complex has been conserved throughout evolution. Tim10p and Tim12p may act akin to molecular chaperones: Tim10p may prevent aggregation of the precursor in the intermembrane space, and Tim12p may stabilize the precursor in a conformation that permits its en bloc insertion (18) into the inner membrane.

  • * To whom correspondence should be addressed. E-mail: schatz{at}ubaclu.unibas.ch

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