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
. 1999 Mar 30;96(7):3634-9.
doi: 10.1073/pnas.96.7.3634.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

T Kanamori  1 , S Nishikawa, M Nakai, I Shin, P G Schultz, T Endo
Affiliations

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

T Kanamori et al. Proc Natl Acad Sci U S A. .

Abstract

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

PubMed Disclaimer

Figures

Figure 1
Figure 1
The DHFR domain of pSu9-DHFR is unfolded at stage B but not at stage A on the mitochondrial surface. pSu9-DHFR was incubated with CCCP-treated mitochondria for 10 min at 0°C (lanes 1–3 and 7–9) or at 30°C (lanes 4–6 and 10–12). After 5-fold dilution with MSC buffer containing 10 mM KCl, 1 μM methotrexate, and 1 mM NADPH, the mitochondria were reisolated by centrifugation and were resuspended in the same buffer. The samples were divided into halves. For lanes 1–6, one aliquot was kept at 0°C (lanes 1 and 4) and the other was treated with 100 μg/ml proteinase K for 15 min at 0°C, which was inactivated by the subsequent addition of 1 mM phenylmethylsulfonyl fluoride (lanes 2, 3, 5, and 6). For lanes 7–12, one aliquot was kept at 0°C (lanes 7 and 10) and the other was treated with 2 μg/ml MPP and 1 mM MnCl2 for 5 min at 30°C, and MPP was inactivated by the subsequent addition of 5 mM EDTA (lanes 8, 9, 11, and 12). The samples were centrifuged, and proteins in the pellet (p) and those in the supernatant (s), which were precipitated with trichloroacetic acid, were analyzed by SDS/PAGE. TEMP, temperature during the binding reaction; p, pellet; s, supernatant; pre and m, precursor and mature forms of pSu9-DHFR, respectively; f, proteinase K-resistant DHFR domain.
Figure 2
Figure 2
Site-specific photocrosslinking reveals interactions between pSu9-DHFR and the TOM components at stages A and B. (A) pSu9-DHFR containing BPA at positions 15, 20, 27, and 108 was bound to CCCP-treated mitochondria for 10 min at 0°C (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or at 30°C (lanes 3, 4, 7, 8, 11, 12, 15, and 16). The mitochondria were reisolated and suspended with MSC buffer containing 10 mM KCl. The samples were divided into halves. One aliquot was subjected to UV irradiation for 5 min at 0°C (even-numbered lanes). Proteins in all the samples were analyzed by SDS/PAGE. Dots indicate the crosslinked products X1–X7, the partners of which are identified as shown on the right side of the gel. Apparent molecular masses of X1, X2, X3, X4, X5, X6, and X7 are 34, 50, 65, 68, 74, 80, and 100 kDa, respectively, on a 10.5% gel, and those of X2, X3, X4, X5, X6, and X7 are 50, 65, 68, 70, 75, and 82 kDa, respectively, on an 8% gel. UV, UV irradiation; BPA, residues at which BPA was introduced; p, pSu9-DHFR. (B) Summary of the results of site-specific photocrosslinking. Crosslinking experiments were performed for the pSu9-DHFR translocation intermediate at stage A or stage B as described in A. The amounts of crosslinked products X1–X7 were quantified and plotted against the positions of introduced BPA (the boxes for the primary structure indicate the positions at which BPA was introduced). Open bars represent the stage A intermediate and solid bars represent the stage B intermediate. The amount of the precursor form of pSu9-DHFR recovered with mitochondria under the same conditions without UV irradiation was set to 100%.
Figure 3
Figure 3
Effects of deletion of the IMS domain of Tom22 on the crosslinking between pSu9-DHFR and the TOM components. (A) Mitochondria were prepared from the yeast MNMS-MAS17 strain (WT) or MNMS-MAS17Δ120–152 strain (ΔC). pSu9-DHFR containing BPA at position 20 or 22 was bound to CCCP-treated mitochondria at 0°C (lanes 1, 2, 5, 6, 9, 10, 13, and 14) or at 30°C (lanes 3, 4, 7, 8, 11, 12, 15, and 16). After binding, the mitochondria were isolated by centrifugation and subjected to UV irradiation for 5 min at 0°C (even-numbered lanes). UV, UV irradiation; MITO, mitochondria; BPA, positions at which BPA was introduced; p, pSu9-DHFR. Triangles indicate the crosslinked product X2 involving Tom22. Dots indicate the crosslinked products X1, X3, X4, and X5. (B) pSu9-DHFR containing BPA at positions 6, 11, 15, 20, 22, 23, 25, 29, 88, 98, and 108 was bound to CCCP-treated mitochondria prepared from MNMS-MAS17 (WT) or from MNMS-MAS17Δ120–152 (ΔC) at 30°C. The samples were diluted with MSC buffer containing 10 mM KCl and divided into halves, and the mitochondria were reisolated by centrifugation. One aliquot was resuspended with MSC buffer containing 10 mM KCl and kept on ice (− chase). The other aliquot was resuspended with chase buffer and incubated for 10 min at 30°C (+ chase). The samples were divided into halves and one aliquot was subjected to UV irradiation for 5 min at 0°C. Proteins in all samples were analyzed by SDS/PAGE, and yields of crosslinked products X1–X7 were quantified as described in Fig. 2.
Figure 4
Figure 4
Chase of pSu9-DHFR from stage B but not from stage A depends on the presence of the IMS domain of Tom22. (A) pSu9-DHFR was bound to CCCP-treated mitochondria prepared from MNMS-MAS17 (WT) or from MNMS-MAS17Δ120–152 (ΔC) at 0°C (lanes 1–8) or at 30°C (lanes 9–16). The samples were divided into four aliquots, which were diluted 5-fold with MSC buffer containing different concentrations of KCl to result in final KCl concentrations of 10 mM KCl (lanes 1, 2, 9, and 10), 50 mM KCl (lanes 3, 4, 11, and 12), 100 mM KCl (lanes 5, 6, 13, and 14), or 150 mM KCl (lanes 7, 8, 15, and 16) and incubated for 5 min at 0°C. The samples were divided into halves and the mitochondria were reisolated by centrifugation. One aliquot was resuspended with binding buffer and kept on ice (odd-numbered lanes). The other aliquot was resuspended with chase buffer and incubated for 10 min at 30°C (even-numbered lanes). p and m, precursor and mature forms of pSu9-DHFR, respectively. (B Upper) Quantification of the bound precursor form (odd-numbered lanes of A). (Lower) Chased mature-sized form (even-numbered lanes of A). The amount of the bound precursor form at 10 mM KCl at 0 or 30°C is set to 100%.
Figure 5
Figure 5
A working model for protein translocation across the outer mitochondrial membrane. The triangle indicates the MPP cleavage site of the fusion protein. Δ, High temperature; IMS, the intermembrane space domain; 22, Tom22; 40, Tom40.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Omura T. J Biochem. 1998;123:1010–1016. - PubMed
    1. Schatz G. J Biol Chem. 1996;271:31763–31766. - PubMed
    1. Neupert W. Annu Rev Biochem. 1997;66:863–917. - PubMed
    1. Pfanner N, Craig E A, Hölinger A. Annu Rev Cell Dev Biol. 1997;13:25–51. - PubMed
    1. Endo T. In: Molecular Chaperones in the Life Cycle of Proteins. Fink A L, Goto Y, editors. New York: Dekker; 1997. pp. 435–466.

Publication types

MeSH terms

Substances

LinkOut - more resources