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Previous Article | Next Article 
Molecular and Cellular Biology, February 2000, p. 1187-1193, Vol. 20, No. 4
Biozentrum, University of Basel, CH-4056
Basel, Switzerland
Received 27 August 1999/Returned for modification 19 October
1999/Accepted 9 November 1999
Import of carrier proteins from the cytoplasm into the
mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and
Tim12p and the integral membrane subunits Tim22p and Tim54p. We
identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form.
When mitochondria are gently solubilized, Tim18p comigrates with the
other subunits of the TIM22 complex on nondenaturing gels and is
coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not
cofractionate with the TIM23 complex upon immunoprecipitation or
nondenaturing gel electrophoresis. Deletion of Tim18p decreases the
growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also
impairs the import of several precursor proteins into isolated
mitochondria, and lowers the apparent mass of the TIM22 complex. We
suggest that Tim18p functions in the assembly and stabilization of the
TIM22 complex but does not directly participate in protein insertion
into the inner membrane.
Most mitochondrial proteins are
synthesized in the cytosol with a cleavable N-terminal presequence that
specifies import into mitochondria via the general protein import
pathway (25, 27, 32, 36). This pathway is mediated by
cytosolic chaperones, a hetero-oligomeric TOM complex in the
mitochondrial outer membrane, a Tim17p-Tim23p complex (referred to as
the TIM23 complex) in the inner membrane, an ATP-dependent import motor
associated with the matrix face of the TIM23 complex, and soluble
proteins in the matrix involved in the proteolytic maturation and
folding of the imported proteins (9, 11, 27-29).
Over the past few years it has become clear that mitochondria possess
an additional pathway which affects import of hydrophobic inner
membrane proteins (17, 22, 27). This pathway diverges from
the general import pathway after the TOM channel (23). As
the hydrophobic precursor exits that channel, it is met by one of the
two soluble 70-kDa protein complexes that transfer it across the
intermembrane space (1, 19, 21, 39). One of these 70-kDa
complexes contains Tim9p and Tim10p, while the other contains Tim8p and
Tim13p (20). Both complexes generally deliver hydrophobic
proteins to an inner membrane complex specialized for the insertion of
membrane proteins. This insertion complex, referred to as the TIM22
complex, has an apparent mass of 300 kDa and contains the membrane
proteins Tim22p and Tim54p, the peripheral membrane protein Tim12p, and
a small proportion of Tim9p and Tim10p (1, 18, 19, 21, 39).
The 70-kDa Tim9p-Tim10p complex can also deliver some membrane proteins
to the TIM23 complex and, possibly, to additional, as-yet-unknown
insertion sites (24).
A typical protein imported by this novel pathway is the ADP/ATP carrier
(AAC), which contains six membrane-spanning regions (7, 30).
AAC lacks a cleavable N-terminal targeting sequence, carrying instead
targeting information in discrete regions throughout the polypeptide
chain (7, 30). Yeast has about three dozen members of the
mitochondrial metabolite carrier family (26). The novel
pathway probably imports all of these, as well as many other integral
proteins of the mitochondrial inner membrane (4, 16, 24).
Here we describe Tim18p, a novel subunit of the TIM22 complex. We
initially identified Tim18p as a protein that coimmunoprecipitated with
Tim54p and then confirmed that it is indeed a subunit of the TIM22
complex. Tim18p is an integral inner membrane protein derived from a
precursor protein of 21.9 kDa which is processed to its mature form
within mitochondria. Its deletion decreases the growth rate of yeast by
half and is synthetically lethal with temperature-sensitive (ts)
mutations in Tim9p or Tim10p. Mitochondria prepared from a Tim18-less
strain are defective in the import of several precursor proteins, have
lowered levels of Tim23p, and contain a TIM22 complex of smaller
apparent size.
Plasmids and strains.
For in vitro transcription and
translation, the DNA fragments encoding Tim23p (15) and
Tim54p were subcloned into pGEM3Z (Promega), the fragment encoding
Tim22p was subcloned into pSP64 (Promega, 19), the
fragments encoding Tim18p and Coq2p and AAC2 were subcloned
into pSP65. The plasmid carrying TIM17 was kindly provided
by Nikolaus Pfanner (University of Freiburg, Freiburg, Germany).
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Tim18p, a New Subunit of the TIM22 Complex That
Mediates Insertion of Imported Proteins into the Yeast Mitochondrial
Inner Membrane
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
tim18 strain.
tim10::HIS3 tim10-1:LEU2. This
strain was mated to the strain lacking Tim18p
(tim18::KANR), the resulting
diploid was allowed to sporulate, and the asci were subjected to tetrad
analysis. A tim9-3 strain was used in which TIM9
had been replaced by TRP1 and the ts tim9-3 allele had been
integrated at the leu2 locus; the resulting strain was tim9::TRP1 tim9-3:LEU2. It was mated to the
strain lacking Tim18p (tim18::KANR), and the diploid was
subjected to tetrad analysis. Viability of dissected spores was tested
at 25°C and auxotrophic markers were screened on the appropriate media.
Coimmunoprecipitation of Tim18p with Tim54p.
Protein
A-Sepharose beads (Pharmacia; 600 mg) were incubated first with bovine
serum albumin and then for 1.5 h with 7.5 ml of anti-Tim54p or
anti-Tim44p rabbit sera made up to a final volume of 15 ml with 50 mM
K+-HEPES (pH 7.4). Beads were washed and incubated with
0.04% glutaraldehyde in 0.1 M sodium phosphate (pH 8.0) for 30 min at
room temperature. After further incubation with 0.2 M Tris-Cl (pH 8.5)
for 1.5 h, followed by a wash, the beads were incubated for 3 min
with 0.1 M glycine-HCl (pH 2.5) and then neutralized with 1 M Tris
base. Mitochondria (26 mg) from the wild-type yeast strain D273-10B were suspended in 3 ml of 0.6 M sorbitol-20 mM K+-HEPES
(pH 7.4) containing 3.4 mg of bovine serum albumin per ml and diluted
with an equal volume of buffer A (20 mM Na+-HEPES [pH
7.0], 30 mM NaCl, 10% glycerol, 20 µM ZnSO4, 1 mM
phenylmethylsulfonyl fluoride [PMSF], 0.5 mM
N
-p-tosyl-L-lysine chloromethyl
ketone [TLCK]). After 15 min on ice, mitochondria were pelleted by
centrifugation, resuspended at 5 mg/ml in buffer A, and preextracted by
mixing with 1 volume of 0.9% n-dodecyl maltoside in buffer
A. After 10 min on ice, insoluble material was pelleted by
centrifugation, resuspended in buffer A to 10 mg/ml, and solubilized
with 1 volume of 3% digitonin in buffer A. After 15 min on ice,
insoluble matter was removed by centrifugation (128,000 × g for 15 min at 2°C), and the supernatant was loaded onto two
35-ml 15 to 25% linear glycerol gradients in buffer A and centrifuged
in a Beckman TST 28.38 ultracentrifuge rotor at 90,000 × g for 16 h at 2°C. Fractions of 1.5 ml were collected, and
10 µl of each was blotted onto a nitrocellulose sheet, which was then
immunoblotted with an antiserum against Tim54p. Fractions enriched for
Tim54p were pooled and used for immunoprecipitation.
20°C overnight. The
precipitated samples were resolved by electrophoresis on a 10%
Tris-tricine gel (35). The gel was stained with Coomassie brilliant blue and, after destaining, the bands of interest were excised and subjected to in-gel tryptic digestion. Peptides were identified by electrospray mass spectrometry (6).
Import of radiolabeled proteins into isolated mitochondria. Mitochondria were purified from lactate-grown yeast cells (13) and assayed for in vitro protein import as described elsewhere (31). Proteins were synthesized in a rabbit reticulocyte lysate in the presence of [35S]methionine after in vitro transcription of the corresponding gene by SP6 or T7 polymerase. The reticulocyte lysate containing the radiolabeled precursor was incubated with isolated mitochondria at the indicated temperatures in import buffer (1 mg of bovine serum albumin per ml, 0.6 M sorbitol, 150 mM KCl, 10 mM MgCl2, 2.5 mM EDTA, 2 mM ATP, 2 mM NADH, 20 mM K+-HEPES [pH 7.4]). Where indicated, the potential across the mitochondrial inner membrane was dissipated with 1 µM valinomycin. Nonimported radiolabeled proteins were removed by treatment with 100 µg of trypsin or 50 µg of proteinase K per ml for 15 to 30 min on ice. Trypsin was inhibited with 200 µg of soybean trypsin inhibitor per ml, and proteinase K was inhibited with 1 mM PMSF. To generate mitoplasts, mitochondria in import buffer were diluted with 9 volumes of 20 mM K+-HEPES (pH 7.4) and incubated at 4°C for 30 min (12). For alkali extraction, mitochondria from an import reaction were sedimented by centrifugation, resuspended to 0.1 mg/ml in 100 mM Na2CO3, and incubated for 30 min at 4°C (8). Supernatant and pellet were separated by centrifugation at 100,000 × g for 15 min.
Blue native gel electrophoresis. Mitochondria (2.5 mg/ml) were solubilized in 20 mM K+-HEPES (pH 7.4)-50 mM NaCl-10% glycerol-2.5 mM MgCl2-1 mM EDTA, plus either 0.16% n-dodecyl maltoside or 1% digitonin (Boehringer Mannheim). After 30 min on ice, insoluble material was removed by centrifugation at 100,000 × g for 10 min, and the solubilized proteins were analyzed by blue native gel electrophoresis on a 6 to 16% linear polyacrylamide gradient (5, 21, 33, 34).
Miscellaneous. An 86-residue C-terminal fragment of Tim54p was expressed in Escherichia coli as a glutathione S-transferase fusion protein and, following cleavage by thrombin and gel purification, was used to raise monospecific antisera in rabbits by standard procedures. Determining the subcellular localization of proteins (12) and coimmunoprecipitation assays (21) were done as described earlier. Mitochondrial membrane potential was determined by the uptake of the lipophilic cation triphenylmethylphosphonium (TPMP) (2). For this assay, 250 µg of mitochondria were incubated in import buffer lacking bovine serum albumin, with 50 nCi of [3H]TPMP per ml and 500 nM unlabeled TPMP for 3 min. They were then sedimented by centrifugation, and [3H]TPMP in the supernatant and pellet were quantified by liquid scintillation counting. The membrane potential was estimated from the accumulation ratio, after subtracting TPMP accumulation measured in a parallel incubation containing 4 µM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), and assuming a mitochondrial volume of 0.7 µl per mg of protein (3). Protein concentration was assayed by the bicinchoninic acid (BCA) method (Pierce) with bovine serum albumin as the standard.
Nucleotide sequence accession number. The nucleotide sequence of the TIM18 gene has been submitted to GenBank under accession number BankIt300568 AF200324.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Identification and localization of Tim18p. To identify novel components of the TIM22 complex, yeast mitochondria were preextracted with n-dodecyl maltoside and then solubilized with digitonin. The solubilized proteins were further separated by centrifugation through a glycerol gradient, and fractions enriched for Tim54p were immunoprecipitated with monospecific antisera against Tim54p. In addition to Tim10p and Tim22p, a band of ~18 kDa was seen that was absent from the control precipitates with nonimmune serum (not shown). Tryptic digestion of this protein band followed by electrospray mass spectrometry identified two peptides (Fig. 1A, boldface letters), whose sequences corresponded to the open reading frame (ORF) yOR297c in the yeast genome that encodes a 21.9-kDa polypeptide of unknown function (Fig. 1A). We designated this ORF TIM18. Its predicted product, Tim18p, has a predicted cleavage site for mitochondrial processing peptidase (RXXS/A [40]) between amino acids 42 and 43 (Fig. 1A, arrow), and three putative transmembrane regions (Fig. 1B). It has significant homology to the succinate dehydrogenase membrane anchor subunit (Sdh4p) of yeast mitochondria (39% identical, 58% similar) and to the putative product of ORF yLR164w (53% identical, 74% similar).
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-ketoglutarate dehydrogenase (KDH), and
cytochrome b2 (Cyt b2)
(Fig. 2A). When the mitochondrial outer
membrane was ruptured by osmotic shock, the tagged Tim18p remained
associated with the mitoplasts, but its C-terminal HA tag became
susceptible to proteolytic digestion (Fig. 2A). Like AAC and Tim54p,
the tagged Tim18p was not extracted by alkali (Na2CO2), indicating that it is an integral
membrane protein (Fig. 2A). To confirm that Tim18p is a mitochondrial
protein, radiolabeled Tim18p was incubated with energized mitochondria.
Tim18p was imported rapidly and processed to its mature form (Fig. 2B).
We conclude that Tim18p is an integral protein of the mitochondrial
inner membrane whose C terminus is exposed to the intermembrane space.
|
Tim18p is a subunit of the TIM22 complex.
To investigate the
role of Tim18p in mitochondrial function, we disrupted the
TIM18 gene. The resulting tim18 strain was viable, but its growth rate on nonfermentable carbon sources was about
half that of the corresponding wild-type strain (not shown). The
tim18 strain had only half as much Tim23p (Fig.
3B) but twice as much Tim12p and Tim10p
as the wild-type strain (Fig. 3A). The levels of Tim54p, Tim22p,
Tim44p, AAC, Cyt b2 and porin were not significantly different from those of the wild-type strain (Fig. 3A).
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tim18 tim10-1 or tim18 tim9-3 meiotic
segregants from the appropriate crosses (Table
1). The genetic interaction between
Tim18p and either Tim9p or Tim10p suggests that Tim18p functions in the
mitochondrial import of inner membrane proteins.
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tim18 in Fig. 4A). The apparent size and
stability of three other high-molecular-weight complexes, including
F1-ATPase, the TOM complex, and cytochrome oxidase, remained unaltered
in these mitochondria (data not shown). This result suggests that
Tim18p is a subunit of the TIM22 complex and that its deletion
specifically impairs the assembly or the stability of this complex.
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Deletion of Tim18p slows import of several mitochondrial precursor proteins. As Tim18p is a subunit of the TIM22 complex, its deletion should affect the import of proteins that require the TIM22 complex for insertion into the mitochondrial inner membrane. We therefore compared the rates of import of several radiolabeled precursor proteins into isolated mitochondria from wild-type cells and from a strain deleted for Tim18p (Fig. 5). As expected, Tim18p-less mitochondria imported the inner membrane proteins Tim23p, Tim22p, Tim54p, and Coq2p two- to threefold more slowly than the corresponding wild-type mitochondria (Fig. 5A to D). To our surprise, however, they imported Tim17p and AAC at close to normal rates (not shown) and imported and processed the matrix-targeted precursors Hsp60 and Su9-DHFR more slowly than did wild-type mitochondria (Fig. 5E and F).
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tim18, 109.7 ± 12.9 mV;
n = 4 to 6). The lowered import rates of precursor
proteins into mitochondria from the Tim18p-less strain are thus not
caused by an indirect effect of a lowered potential across the
mitochondrial inner membrane.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have identified Tim18p as a new subunit of the TIM22 complex that mediates insertion of hydrophobic proteins into the mitochondrial inner membrane. This identification rests on three observations. First, Tim18p is coimmunoprecipitated with Tim22p, Tim54p, and Tim12p in the presence of nondenaturing detergents. Second, it comigrates with Tim22p and Tim54p as a 300-kDa complex upon blue native gel electrophoresis. Third, its deletion affects the apparent size of the TIM22 complex. Furthermore, Tim18p is not associated with other components of the mitochondrial protein import machinery such as the TIM23 complex.
This brings the number of known subunits of the TIM22 complex to six: Tim22p (38), Tim12p and Tim10p (19, 39), Tim54p (18), Tim9p (1, 20), and Tim18p (this study).
Tim18p not only is physically associated with the TIM22 complex but also is required for the optimal function of that complex. Deletion of TIM18 is synthetically lethal with ts mutations in either TIM9 or TIM10, whose protein products are components of both the TIM22 complex and the 70-kDa complex in the soluble intermembrane space (1, 19, 21, 39). Deletion of Tim18p also slows import of several precursor proteins into isolated mitochondria.
Deletion of Tim18p impairs import of several inner membrane proteins, including Tim22p, Tim54p, and Coq2p by two- to threefold, yet it does not lower the steady-state levels of these proteins. The slower import of Tim23p into Tim18p-less mitochondria agrees with the findings that this import is mediated by the TIM22 complex (4, 16) and that Tim18p-less mitochondria have only half as much Tim23p as do wild-type mitochondria (Fig. 3B). As Tim23p mediates import of proteins into the matrix, the decreased import and processing of matrix-targeted precursors into Tim18p-less mitochondria is thus probably a secondary consequence of the decreased abundance of Tim23p. No such defect has been reported for mutations in other components of the TIM22 complex. However, it is also possible that deletion of Tim18p affects the activity of the TIM23 complex by a more direct mechanism. It is puzzling that deletion of Tim18p affects neither import rates nor steady-state levels of AAC, even though AAC import requires the TIM22 complex. Perhaps Tim18p is not essential for the ability of the TIM22 complex to insert AAC into the mitochondrial inner membrane.
The precise role of Tim18p in the functioning of the TIM22 complex remains to be established. Tim18p probably does not bind the imported precursors directly, since we were unable to cross-link radiolabeled Tim23p to Tim18p in isolated mitochondria (not shown). The fact that Tim18p is not essential for viability, even though its deletion decreases the apparent size of the TIM22 complex, suggests that Tim18p may be involved in the assembly or the stabilization of the TIM22 complex rather than in its function. Further analysis will provide insights into how Tim18p cooperates with the known subunits of the TIM22 complex, with perhaps additional, as-yet-unknown subunits and with the two soluble 70-kDa complexes in the intermembrane space to insert hydrophobic proteins into the mitochondrial inner membrane.
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ACKNOWLEDGMENTS |
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C. M. Koehler and M. P. Murphy contributed equally to this paper.
We are grateful to Nikolaus Pfanner for his generous gift of the plasmid carrying the TIM17 gene and to Paul Jeno for assistance with the electrospray mass spectrometry and peptide sequencing.
We thank the following agencies for their support: the Swiss National Science Foundation (to G.S.), the Human Frontier Science Program Organization (to G.S.), the European Economic Union (to G.S.), the F. Louis Jeantet Foundation (to G.S.), the Damon Runyon-Walter Winchell Cancer Research Foundation (to C.M.K.), the U.S. National Science Foundation (to C.M.K.), and the European Molecular Biology Organisation (to E.O.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Cell Biology, Harvard Medical School, 25 Shattuck St., Boston, MA 02115. Phone: (617) 432-1611. Fax: (617) 432-1190. E-mail: eran_or{at}hms.harvard.edu.
Present address: Department of Chemistry and Biochemistry,
University of California
Los Angeles, Los Angeles, CA 90095-1569.
Present address: Department of Biochemistry, University of Otago,
Dunedin, New Zealand.
§ Retired.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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). After import,
samples were treated with proteinase K to remove nonimported precursor
and further processed as described previously (
-particle emissions (103) for Tim23p were quantified by
using a 
