Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Apr;21(7):2337-48.
doi: 10.1128/MCB.21.7.2337-2348.2001.

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors

C Meisinger  1 , M T Ryan, K Hill, K Model, J H Lim, A Sickmann, H Müller, H E Meyer, R Wagner, N Pfanner
Affiliations

Protein import channel of the outer mitochondrial membrane: a highly stable Tom40-Tom22 core structure differentially interacts with preproteins, small tom proteins, and import receptors

C Meisinger et al. Mol Cell Biol. 2001 Apr.

Abstract

The preprotein translocase of the yeast mitochondrial outer membrane (TOM) consists of the initial import receptors Tom70 and Tom20 and a approximately 400-kDa (400 K) general import pore (GIP) complex that includes the central receptor Tom22, the channel Tom40, and the three small Tom proteins Tom7, Tom6, and Tom5. We report that the GIP complex is a highly stable complex with an unusual resistance to urea and alkaline pH. Under mild conditions for mitochondrial lysis, the receptor Tom20, but not Tom70, is quantitatively associated with the GIP complex, forming a 500K to 600K TOM complex. A preprotein, stably arrested in the GIP complex, is released by urea but not high salt, indicating that ionic interactions are not essential for keeping the preprotein in the GIP complex. Under more stringent detergent conditions, however, Tom20 and all three small Tom proteins are released, while the preprotein remains in the GIP complex. Moreover, purified outer membrane vesicles devoid of translocase components of the intermembrane space and inner membrane efficiently accumulate the preprotein in the GIP complex. Together, Tom40 and Tom22 thus represent the functional core unit that stably holds accumulated preproteins. The GIP complex isolated from outer membranes exhibits characteristic TOM channel activity with two coupled conductance states, each corresponding to the activity of purified Tom40, suggesting that the complex contains two simultaneously active and coupled channel pores.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Stability of the 400K GIP complex. (A) Mitochondria (100 μg of protein) were lysed in solubilization buffer containing 1% digitonin and the indicated concentration of urea for 10 min on ice. After a clarifying spin, protein complexes were separated by BN-PAGE and blotted onto PVDF membranes, followed by immunodecoration using antisera against Tom40, Tom22, and porin. As control, 25% of the samples were directly subjected to SDS-PAGE and immunodecoration. (B) Mitochondria (100 μg of protein) were resuspended in 100 mM Na2CO3 (pH 11.5) and incubated for 30 min on ice. After centrifugation at 100,000 × g, the nonextractable pellet was lysed in digitonin buffer and subjected to BN-PAGE, followed by immunodecoration against Tom40 and Tom22 (lane 1); the control represents mitochondria without carbonate treatment. A control sample, the nonextractable pellet, and the TCA-precipitated supernatant (Sn.) were also analyzed by SDS-PAGE, showing the correct fractionation patterns (lanes 6 to 8). BN-PAGE of control samples and the carbonate-resistant fractions, followed by Western blot analysis using antisera against Tim22, Tim23, the β subunit of the F1-ATPase, and Hsp60, revealed no carbonate-resistant complexes (lanes 9 to 12). For salt extraction (lanes 3 to 5), mitochondria were resuspended and incubated in SEM buffer containing the indicated NaCl concentrations for 10 min on ice. After the salt was washed off with SEM buffer, the mitochondria were lysed in digitonin-containing buffer and subjected to BN-PAGE.
FIG. 2
FIG. 2
Association of the small Tom proteins with the 400K GIP complex. (A) Stability of the small Tom proteins in the 400K complex following urea treatment. Mitochondria (50 μg of protein) were incubated with the radiolabeled preproteins of the small Tom proteins in import buffer for 10 min at 25°C. After lysis in solubilization buffer containing 1% digitonin and the indicated concentrations of urea, the samples were subjected to BN-PAGE and digital autoradiography. (B) Quantitation of the urea-resistant Tom proteins in the 400K complex (as described for Fig. 1A and panel A). The total amount of each Tom protein in the 400K complex in the absence of urea was set to 100% (control). (C) Carbonate resistance of the small Tom proteins in the 400K complex. Radiolabeled preproteins of the small Tom proteins were imported into mitochondria (50 μg of protein) for 10 min at 25°C. Mitochondrial pellets were resuspended in 100 mM Na2CO3 (pH 11.5) and incubated on ice for 30 min. After centrifugation at 100,000 × g for 30 min, the carbonate-resistant pellets were solubilized in 1% digitonin and separated by BN-PAGE (lanes 4 to 6). As a control, mitochondria containing imported Tom proteins were directly lysed and separated by BN-PAGE (lanes 1 to 3). Tom40 was detected by immunodecoration in a control sample and a carbonate-resistant pellet (lanes 7 and 8, respectively). The amount of carbonate-resistant Tom protein was quantified and compared to the total amount of the respective Tom protein in the 400K complex (control) (columns 9 to 12). For comparison, radiolabeled small Tom proteins were imported into mitochondria (lane 13), separated into carbonate-resistant pellet (lane 14) and carbonate-extractable supernatant (Sn.; TCA precipitated; lane 15), and subjected to SDS-PAGE.
FIG. 3
FIG. 3
Relation of Tom20 and Tom70 to the yeast GIP complex. (A) BN-PAGE of mitochondria (50 μg of protein) lysed in different concentrations of digitonin, followed by immunodecoration with Tom40 antiserum. The high-molecular-weight complex observed at 0.1% digitonin is indicated by an asterisk. (B) Two-dimensional electrophoresis, BN-PAGE followed by SDS-PAGE, of mitochondria (100 μg of protein) solubilized in 1% digitonin (top) or 0.1% digitonin (bottom). As a control, mitochondria (20 μg of protein) were loaded directly onto the SDS-PAGE and electrophoresed in one dimension (bottom, Mito control). Immunodecoration was performed with antiserum directed against Tom70, Tom40, Tom20, or Tom5. (C) Binding of Tom proteins from Tom22-His mitochondria (left) or wild-type mitochondria (right) onto Ni-NTA. Mitochondrial membranes were isolated and solubilized as described in Materials and Methods. After binding and collection of the flowthrough fraction, the column was washed with 50 mM imidazole followed by 80 mM imidazole, and the TOM complex was eluted at 200 mM imidazole. Aliquots of each fraction were subjected to SDS-PAGE and immunoblotting.
FIG. 4
FIG. 4
Accumulation and stability of a preprotein in the GIP complex. (A) Schematic diagram depicting the experimental procedures for analysis of AAC-DHFR arrested in the TOM complex. MTX, methotrexate. (B) Arrested AAC-DHFR dissociates from the complex in the presence of urea but not high salt concentrations. Radiolabeled AAC-DHFR was incubated with mitochondria (50 μg of protein) in the presence of methotrexate for 25 min at 25°C. The mitochondria were either treated with NaCl (as indicated) in SEM buffer followed by solubilization in 1% digitonin or directly solubilized in digitonin in the presence of the indicated urea concentrations. After BN-PAGE, AAC-DHFR was detected by digital autoradiography. (C) Quantitation of GIP-arrested AAC-DHFR after high-salt (top) or urea (bottom) treatment. In the lower graph, the quantitation of proteinase K-resistant DHFR after treatment with urea is also shown (see Materials and Methods). (D) The small Tom proteins are not needed to keep a preprotein in the TOM complex. Mitochondria containing arrested radiolabeled AAC-DHFR were solubilized in either 1% digitonin or 0.5% Triton X-100 and subjected to BN-PAGE (lanes 2 and 3). The TOM complex lacking arrested preprotein is shown in lanes 4 and 5. The pellet arising from carbonate-treated mitochondria containing arrested AAC-DHFR was solubilized in 1% digitonin (lane 1). Mitochondria containing imported small Tom proteins were lysed in 1% digitonin or 0.5% Triton X-100 (lane 6 to 11). The blue native gels were blotted onto PVDF membranes, and radiolabeled proteins were detected by digital autoradiography. Lanes 4 and 5 were immunodecorated with Tom40 antiserum. (E) The amount of individual Tom proteins retained in the complex following Triton X-100 solubilization and BN-PAGE (as described for panel D) were quantitated and compared to the amount of protein found in digitonin-lysed mitochondria (set to 100%).
FIG. 5
FIG. 5
Accumulation of a preprotein in purified outer membrane vesicles. (A) Purity of outer membrane vesicles. Mitochondria (Mitoch.; 25 μg of protein) and outer membrane vesicles (2.5 μg of protein) were separated by SDS-PAGE, blotted on PVDF membranes, and probed with antisera against proteins of the outer membrane (Tom40 and porin), the inner membrane (Tim23), and the intermembrane space (Tim12 and Tim10). (B) Stability of the 400K GIP complex of outer membrane vesicles against treatment with urea. Outer membrane vesicles (5 μg of protein) were lysed in 1% digitonin in the presence of the indicated urea concentrations and subjected to BN-PAGE, followed by immunodecoration of Tom40. (C) Accumulation of AAC-DHFR in outer membrane vesicles. Radiolabeled AAC-DHFR was imported into outer membrane vesicles (5 μg of protein; lanes 1 to 4) or mitochondria (50 μg of protein; lanes 5 to 8) in the presence or absence of methotrexate (MTX). Where indicated, the outer membrane vesicles and the mitochondria were pretreated with trypsin prior to the import reaction to remove the surface receptor domains.
FIG. 6
FIG. 6
The GIP complex eluted from outer membrane vesicles after BN-PAGE is functional. (A) Schematic diagram showing isolation of the 400K GIP complex. (B and C) Current traces from a bilayer fused with 400K complex containing liposomes under symmetric conditions (250 mM KCl, 10 mM MOPS-Tris [pH 7.0] on both sides of the membrane). The bottom trace of panel C shows a time scale-expanded current recording with high time resolution. (D) Current-voltage relationship of the two most frequent conductance states with the same symmetric KCl concentrations as in panel B. (E) Current-voltage relationship of the main conductance level at asymmetric buffers (cis, 250 mM KCl; trans, 20 mM KCl) for determination of reverse potential.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahting U, Thun C, Hegerl R, Typke D, Nargang F E, Neupert W, Nussberger S. The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J Cell Biol. 1999;147:959–968. - PMC - PubMed
    1. Alconada A, Gärtner F, Hönlinger A, Kübrich M, Pfanner N. Mitochondrial receptor complex from Neurospora crassa and Saccharomyces cerevisiae. Methods Enzymol. 1995;260:263–286. - PubMed
    1. Alconada A, Kübrich M, Moczko M, Hönlinger A, Pfanner N. The mitochondrial receptor complex: the small subunit Mom8b/Isp6 supports association of receptors with the general insertion pore and transfer of preproteins. Mol Cell Biol. 1995;15:6196–6205. - PMC - PubMed
    1. Bauer M F, Hofmann S, Neupert W, Brunner M. Protein translocation into mitochondria: the role of TIM complexes. Trends Cell Biol. 2000;10:25–31. - PubMed
    1. Blom J, Kübrich M, Rassow J, Voos W, Dekker P J T, Maarse A C, Meijer M, Pfanner N. The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane. Mol Cell Biol. 1993;13:7364–7371. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources