Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3G 1Y6
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Abstract |
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Insertion of newly synthesized proteins into
or across the mitochondrial outer membrane is initiated
by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into
a preprotein translocation pore, comprised of Tom40.
Here, we show that a prominent -barrel channel protein spanning the outer membrane, human voltage-
dependent anion-selective channel (VDAC), bypasses
the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore
with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant
of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently
insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms
newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.
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Introduction |
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SORTING of newly synthesized proteins to specific organelles within the cell requires a mechanism to direct the protein to its correct location and to overcome the thermodynamic barrier imposed by the lipid
bilayer during transmembrane translocation. Typically,
recognition is provided by organelle-specific receptors and
translocation is mediated by a transbilayer machinery that includes a pore complex through which the polypeptide
can move (Schatz and Dobberstein, 1996). In the case of
mitochondria, both the outer and inner membranes are
competent for protein translocation and achieve this by
distinct translocation machineries (Hauke and Schatz,
1997; Neupert, 1997
; Pfanner and Meijer, 1997
). A complex of receptors embedded in the outer membrane
(Tom20, Tom22, Tom37, and Tom70) mediates recognition of all known proteins destined for internalization into
the organelle. After binding to the receptor complex, the
preprotein moves into a translocation apparatus comprised of the predicted channel, Tom40, and the ancillary
proteins, Tom5, Tom6, and Tom7. Recent reconstitution
of functional protein translocation in synthetic lipid bilayers faithfully recapitulates this process and suggests the existence of a transbilayer water-filled pore as the polypeptide translocator (Hill et al., 1998
; Kunkele et al., 1998
).
Mitochondrial preproteins that contain hydrophobic
membrane-spanning segments are likely arrested and released from the translocation pore into the surrounding
lipid bilayer during the translocation process. Such a
mechanism is well documented in the ER (Rapoport et al.,
1996). Indeed, hydrophobic sequences analogous to ER
stop-transfer and signal-anchor sequences have been identified as topogenic sequences that trigger protein integration into the mitochondrial outer membrane (Nguyen et al.,
1988
; Li and Shore, 1992
; McBride et al., 1992
). In contrast, little is understood about
-barrel proteins, prototypically represented by the bacterial porins (Weiss et al.,
1991
), which lack uniformly hydrophobic domains, but
whose overall amphiphilic character permits integration of
almost the complete protein structure into the bilayer to
form a transmembrane channel. Such a porin exists as an
abundant 30-kD voltage-dependent anion-selective channel (VDAC)1 in the mitochondrial outer membrane of all
eukaryotes examined (Colombini et al., 1996
; Mannella,
1997
). It is comprised of a 12- to 13-strand barrel (Colombini et al., 1987
; Stanley et al., 1995
) that provides the major pathway for transport of metabolites, including ATP,
through the membrane (Rostovtseva and Colombini, 1997
;
Lee et al., 1998
). Coordinated regulation of this outer membrane channel with those in the inner membrane may play
an important role in mitochondria function and signaling,
including events contributing to the mitochondrial involvement in apoptosis (Green and Reed, 1998
).
Here, we have examined the import pathway of VDAC.
Tom20 plays a direct role in targeting newly synthesized
VDAC for integration into the mitochondrial outer membrane. Import is blocked by Tom20 gene deletion in vivo
(Sollner et al., 1989) and by competing anti-Tom20 antibodies in vitro (Ramage et al., 1993
; Goping et al., 1995
).
Moreover, a direct physical interaction between VDAC and Tom20 has been observed (Schleiff et al., 1997
). Consistent with this, targeting sequences that specify protein
translocation into or across the outer membrane effectively compete for VDAC insertion into the outer membrane (Millar and Shore, 1996
). We now demonstrate,
however, that VDAC bypasses the requirement for the
Tom40 preprotein translocation pore. Rather, Tom20
alone is capable of catalyzing direct insertion of this
-barrel protein into the membrane lipid bilayer.
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Materials and Methods |
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General
Previous articles (McBride et al., 1992, 1996
; Millar et al., 1996; Goping
et al., 1998
) describe the routine procedures used in this study. These include isolation of rat heart mitochondria, transcription-translation of plasmids encoding VDAC and preornithine carbamyl transferase (pOCT),
and import of 35S-labeled translation products into mitochondria in vitro.
Additional details are provided in the figure legends.
Recombinant Cytosolic Domain of Human Tom20
Recombinant glutathione S-transferase (GST)-human Tom201-29 (formerly named GST-
30hTom20), lacking the NH2-terminal transmembrane segment of hTom20, was expressed in TOPP2 cells and purified
(Schleiff et al., 1997
). Protein immobilized on glutathione-Sepharose 4B was washed and suspended in 20 mM NaPO4, pH 7.4, 150 mM NaCl, 1 mM
dithiothreitol, 0.5 mM CaCl2, and incubated with 5 µg/ml thrombin for 18 h
at 4°C, generating hTom20
1-29 with an extra gly residue at the NH2 terminus derived from the thrombin cleavage site. The mixture was passed
through a Mono S HR 5/5 column and protein was eluted with a linear
gradient (buffer A, 10 mM MES, pH 5.0; buffer B, 10 mM 3-cyclohexylamino-1-propane sulfonic acid, pH 10, 500 mM NaCl). Peak fractions
containing hTom20
1-29 were resolved on a Superdex 200 column in
10 mM Hepes, pH 7.0, and 100 mM NaCl. Fractions containing the purified protein were dialyzed against the same buffer containing 5 mM DTT
and the protein concentrated to 1.5 mg/ml.
Plasmid encoding GST-hTom201-29 was manipulated by standard recombinant DNA procedures to substitute cys at hTom20 codon position
100 with ser and to introduce ser-cys after gly at the thrombin cleavage
site. The thrombin cleavage product, hTom20
1-29/N-GSC/C100S, was
generated and purified as above.
Liposomes
Approximately 100 nm large unilamellar vesicles (LUVs) (lipid composition: PC, phosphotidylcholine; PE, phosphotidylethanolamine; PI, phosphotidylinositol; PS, phosphotidylserine; PC/PE/PI/PS at molar ratios of
55:28:13:3 or PC/PE/PI/PS/PE-bmps at molar ratios of 54.5:27.5:13:3:1)
were prepared in 170 mM sucrose, 20 mM Tris acetate, pH 7.0, and 2 mM
CaCl2 (Shahinian and Silvius, 1995). LUVs containing
-maleimidopropionic acid N-hydroxysuccinimide ester of PE (PE-bmps) were incubated
for 12 h at 4°C with hTom20
1-29/N-GSC/C100S (10-fold molar excess
relative to PE-bmps; coupling efficiency, 85-95%) followed by two 30 min incubations with excess LUVs generated in medium lacking sucrose
(100 mM NaCl, 20 mM Tris acetate, pH 7.0, and 2 mM CaCl2) to competitively remove unincorporated hTom20. Sucrose-loaded LUVs containing
the covalently attached cytosolic domain of hTom20 were recovered by
centrifugation at 50,000 × g for 60 min in a Beckman 75Ti rotor. They
were used in protein import reactions (Goping et al., 1998
) in place of mitochondria.
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Results and Discussion |
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Standard protein import was conducted by combining rat
heart mitochondria with human VDAC synthesized in
reticulocyte lysate. In this system, the preprotein translocation pore can be jammed by partially importing a chimeric protein containing a matrix-targeting signal fused to
dihydrofolate reductase (DHFR). Unfolding of the DHFR
moiety on the cytosolic side of the outer membrane is prevented by the high-affinity DHFR active site inhibitor,
methotrexate (MTX; Eilers and Schatz, 1986; Vestweber
et al., 1989
). In the presence of MTX, excess pODHFR
(Sheffield et al., 1990
), a chimeric protein consisting of the
matrix targeting signal of pOCT fused to DHFR and purified from expressing bacteria, blocked import and processing of pOCT (Fig. 1 A, lower panel, lanes 3 and 4), whose
processed form otherwise acquires resistance to external trypsin (Fig. 1 A, lower panel, lanes 8 and 9). In addition
to inhibition of pOCT import, pODHFR/MTX inhibited
outer membrane insertion of yTom70(1-29)DHFR (Fig. 1
B), also designated pOMD29, a chimeric protein comprised of the transmembrane signal-anchor domain of
yeast Tom70 fused to DHFR, whose insertion into the outer membrane of heart mitochondria in vitro has been
documented previously (Li and Shore, 1992
; McBride et
al., 1992
). yTom70(1-29)DHFR is inserted into the outer
membrane in the Nin-Ccyto orientation (Li and Shore, 1992
;
McBride et al., 1992
) and therefore, its import in the absence of competing pODHFR was unaffected by MTX (not shown).
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In contrast to pOCT and yTom70(1-29)DHFR, import
and membrane insertion of VDAC in this system, assessed
by the temperature-sensitive acquisition of resistance to
alkaline extraction (Fujiki et al., 1982; Goping et al., 1998
;
Fig. 1 A, upper panel, compare lanes 7 and 8), was relatively unaffected by pODHFR/MTX (Fig. 1 A, upper panel, lanes 8 and 9). The slight reduction in VDAC import at 5 min effected by pODHFR/MTX may relate to
the fact that preproteins like pODHFR can stimulate
recruitment of Tom20 into the translocation complex,
thereby competitively reducing the amount of free Tom20 in the membrane that is available for interaction with
VDAC (Rapaport et al., 1998
). The failure of partially
translocated pODHFR/MTX to block membrane insertion of VDAC suggested that VDAC may bypass the requirement for the Tom40 translocation pore during import.
To address this question further, import of VDAC was
examined using isolated yeast mitochondria containing a
temperature-sensitive mutant of Tom40 (Kassenbrock et al.,
1995). In contrast to control mitochondria, temperature-sensitive Tom40 mitochondria were incapable of importing the matrix preprotein form of cytochrome oxidase subunit Va (COX Va) at the nonpermissive temperature
(37°C), as judged by the failure of pCOX Va to be processed by the mitochondria at 37°C (Fig. 1 C, lane 4), while
processing of pCOX Va was observed at the permissive
temperature of 23°C. In contrast to pCOX Va, membrane
insertion of VDAC occurred at both 23 and 37°C (Fig. 1
C). The slight decrease in membrane insertion of VDAC
at 37°C compared with 23°C was similar for both wild-type
and temperature-sensitive Tom40 mitochondria (Fig. 1 C),
suggesting that this difference was related to events other
than the temperature-sensitive phenotype of Tom40.
The cytosolic domain of hTom20, hTom201-29, when
included as a soluble entity in excess in the import reaction, inhibited import of pOCT (Fig. 1 A, lower panel,
compare lanes 8 and 10), presumably because it sequestered the preprotein through direct protein interaction
(Schleiff et al., 1997
) and prevented transfer of the preprotein to the translocation machinery. hTom20
1-29 can
also physically interact with VDAC (Schleiff et al., 1997
). In distinct contrast to pOCT, however, hTom20
1-29 did
not interfere with insertion of VDAC into the outer membrane. In fact, it had a slight stimulatory effect (Fig. 1 A,
upper panel, compare lanes 8 and 10), suggesting that
potential interactions between VDAC and hTom20
1-29
in the import reaction must be readily reversible. Moreover, the difference in response of pOCT and VDAC to
hTom20
1-29 in the import reaction suggested a potential
fundamental difference in the import pathways of the two
proteins. However, the results also imply that a complex of
VDAC and soluble hTom20
1-29 can make contact with
the mitochondrial surface in vitro, a suggestion that is
compatible with the observed binding of the cytosolic domain of hTom20 to lipid surfaces in vitro (Schleiff and
Turnbull, 1998
; Fig. 2 A).
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To assess the possibility that hTom20 can mediate direct
insertion of VDAC into a membrane lipid bilayer, synthetic LUVs were created in which the cytosolic domain of
hTom20 was linked to the bilayer surface via covalent attachment to PE-pmbs (Fig. 2 A). To that end, the protein
was modified to contain a unique cys at the NH2 terminus
of hTom201-29; the other cys located at codon position
100 was converted to ser. These changes did not influence
the ability of the receptor to interact with either VDAC or
pOCT in vitro (data not shown). The liposomes had a
phospholipid composition similar to that of total mitochondrial outer membrane in rat liver (de Kroon et al.,
1997
). Under the conditions used for mitochondrial protein import reactions with VDAC synthesized in reticulocyte lysate such liposomes were found to efficiently insert
the protein, as determined by acquired resistance to extraction at alkaline pH or with urea (Fig. 2 B, upper panel,
lanes 2 and 3). Moreover, the resulting import product was
resistant to treatment with trypsin (lane 4), reflective of
VDAC in native membranes (Colombini et al., 1996
; Mannella, 1997
). LUVs bearing the hTom20 cytosolic domain
did not translocate pOCT, as judged by the failure of
pOCT to acquire resistance to trypsin (Fig. 2 B, lower
panel), nor did they insert yTom70(1-29)DHFR, as judged
by its extractability from liposomes with 7 M urea (not
shown). Conversely, LUVs lacking the hTom20 cytosolic
domain did not insert VDAC (Fig. 2 B, upper panel, lanes
6-8). Furthermore, VDAC that associated with LUVs lacking the hTom20 cytosolic domain exhibited facile interliposomal transfer (Fig. 2 C), indicating that this binding by VDAC was merely peripheral. As expected, VDAC
that was inserted into the LUV lipid bilayer by the
hTom20 cytosolic domain did not transfer to subsequently
added protein-free LUVs. In contrast, pOCT that bound
to LUVs containing the hTom20 cytosolic domain could
subsequently transfer to protein-free LUVs, but slower
than pOCT that bound to LUVs lacking hTom20 (~t1/2, 15 versus 1 min; Fig. 2 C). This finding is consistent with
the ability of hTom20 to physically interact with pOCT
(Schleiff et al., 1997
). Moreover, the relatively slow dissociation of pOCT from the hTom20 cytosolic domain explains the ability of excess hTom20
1-29 to inhibit import
of pOCT into mitochondria in vitro (Fig. 1 A).
Finally, VDAC that had been inserted into LUVs by the
hTom20 cytosolic domain was examined for its ability to
transport a physiological substrate of this channel protein,
ATP (Rostovtseva and Colombini, 1997; Lee et al., 1998
).
LUVs were loaded with [32P]ATP before the import reaction, and the subsequent release of ATP was measured at
various times after the initiation of the reaction. Insertion
of a single functional VDAC channel into the vesicle
would be predicted to release encapsulated ATP. Release of radioactive ATP commenced immediately upon initiation of VDAC insertion into liposomes (Fig. 3). Egress of
ATP was dependent on functional VDAC, inhibited by a
known antagonist, NADH (Zizi et al., 1994
), and required
the presence of the hTom20 cytosolic domain on the liposome surface to integrate VDAC into the lipid bilayer. In
contrast, the control protein pOCT did not stimulate ATP
export from LUVs alone or LUVs with hTom20 (Fig. 3).
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The first indication that a -barrel protein like VDAC
might not require a complex preprotein translocation machinery for insertion into a membrane lipid bilayer came
with the observation that purified detergent-solubilized
VDAC can spontaneously integrate into planar lipid bilayers as a functional entity. Moreover, in this system insertion
is cooperative as a result of self-mediated stimulation (Xu
and Colombini, 1996
). Although the lag period for this insertion was relatively long, the results suggest an inherent
ability of the molecule to partition into a lipid bilayer, but
presumably this is because the detergent-extracted entity is in a favorable state for lipid bilayer integration. However, in normal physiology, other considerations apply: the
question of membrane specificity; the problem of competing interactions of VDAC with other proteins, specific or
otherwise; the necessity to maintain a conformation of soluble VDAC after release from the ribosome that is compatible with subsequent membrane insertion; and the requirement for a mechanism to catalyze VDAC insertion upon contact with the mitochondrial outer membrane. In
the import reaction documented here, the appropriate
translocation-competent conformation for VDAC may be
supplied by chaperones/factors present in reticulocyte lysate (Smith et al., 1994
). The only other minimum requirement was provided by hTom20 on the surface of the membrane. While it is impossible to rule out any involvement
of the Tom40 translocation pore, its contribution to
VDAC membrane insertion was not detected in the assays
described here, whereas its influence on import of matrix
pOCT and pCOX Va, and on membrane insertion of outer
membrane Tom70(1-29)DHFR was pronounced. In addition to its role as a receptor in directing newly synthesized
VDAC exclusively to the mitochondrial outer membrane
within the context of a whole cell, our results suggest that
hTom20 is capable of catalyzing direct insertion of the
protein into the lipid bilayer. The receptor may accomplish this simply by bringing VDAC into close proximity
to the bilayer and/or by triggering release of presumptive
chaperones or other factors from VDAC that otherwise maintain the protein water-soluble. In either scenario,
VDAC may spontaneously insert into the proximate bilayer. Alternatively, or in addition, hTom20 may play a direct role in guiding the conformational change that allows
the transbilayer
-barrel channel to form.
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Footnotes |
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Address correspondence to Gordon C. Shore, Department of Biochemistry, McIntyre Medical Sciences Building, McGill University, 3655 Drummond Street, Montreal, Quebec, Canada H3G 1Y6. Tel.: (514) 398-7282. Fax: (514) 398-7384. E-mail: shore{at}med.mcgill.ca
Received for publication 21 January 1999 and in revised form 20 April 1999.
This work was financed by operating grants from the Medical Research
Council and National Cancer Institute of Canada.
We thank Rania Leventis for help preparing liposomes and Nancy Martin for providing yeast strain KKY3.3.
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Abbreviations used in this paper |
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DHFR, dihydrofolate reductase;
GST, glutathione S-transferase;
hTom20, human Tom20;
LUVs, large unilamellar liposomes;
MTX, methotrexate;
pCOX Va, precytochrome oxidase
subunit Va;
PE, phosphatidylethanolamine;
PE-pmbs, -maleimidopropionic acid N-hydroxysuccinimide ester of PE;
pOCT, preornithine carbamyl transferase;
VDAC, voltage-dependent anion-selective channel.
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