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 Oct 12;96(21):11770-5.
doi: 10.1073/pnas.96.21.11770.

Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b(2) import pathway

M Esaki  1 , T Kanamori, S i Nishikawa, T Endo
Affiliations

Two distinct mechanisms drive protein translocation across the mitochondrial outer membrane in the late step of the cytochrome b(2) import pathway

M Esaki et al. Proc Natl Acad Sci U S A. .

Abstract

The import of cytochrome b(2) into mitochondria consists of two steps. The translocation of the first part of the presequence across the inner membrane is coupled with the translocation of the tightly folded heme-binding domain across the outer membrane and requires a membrane potential DeltaPsi and the functions of mitochondrial Hsp70 (mHsp70) in the matrix. Once the heme-binding domain has passed the outer membrane, the translocation of the rest of the polypeptide chain across the outer membrane becomes independent of DeltaPsi and mHsp70. Here we analyzed the late DeltaPsi- and mHsp70-independent step in the transport of cytochrome b(2) fusion proteins into the intermembrane space (IMS). The import of the cytochrome b(2) fusion proteins containing two protein domains linked by a spacer segment into mitochondria was arrested at a stage at which one domain folded on each side of the outer membrane, along the pathway that is consistent with the stop-transfer model. The mature-size form of the translocation intermediate could move across the outer membrane in both directions, and the stabilization of the protein domain in the IMS promoted the forward translocation. On the other hand, the intermediate-size form of the translocation intermediate, which retains the anchorage to the inner membrane, was transported into the IMS independently of the stability of the protein domain in the IMS. These results suggest that two distinct mechanisms, the Brownian ratchet and the anchor diffusion mechanisms, can operate for the transmembrane movement of the mature-size form and the intermediate-size form, respectively, of cytochrome b(2) species.

PubMed Disclaimer

Figures

Figure 1
Figure 1
(A) pb2(220)-DHFR fusion proteins. (B) A proteinase K digestion of the pb2(220)-DHFR fusion proteins. pb2(220)WT-DHFR (WT), pb2(220)dc1-DHFR (dc1), and pb2(220)dc2-DHFR (dc2) synthesized in vitro were diluted 100-fold with 250 mM sucrose, 10 mM Mops⋅KOH (pH 7.2), 80 mM KCl, 2 mM cold methionine, and 4 μM hemin and were treated with various concentrations of proteinase K (prot K) for 10 min at 25°C. The reactions were stopped by the addition of 1 mM phenylmethylsulfonyl fluoride, and proteins were precipitated by trichloroacetic acid and were analyzed by SDS/PAGE and radioimaging.
Figure 2
Figure 2
In vitro import of pb2(220)WT-DHFR into mitochondria. (A) Radiolabeled pb2(220)WT-DHFR was incubated with isolated yeast mitochondria at 25°C for indicated times. The mitochondria were treated with (+ Prot K, lanes 1–8) or without (− Prot K, lanes 9–16) 100 μg/ml proteinase K for 30 min on ice. (B) Amounts of the total mature-size form (A, lanes 1–8, open squares), those of the protease-protected mature-size form (A, lanes 9–16, open circles), which received the Imp1p processing and was already localized in the IMS, and those of the protease-sensitive mature-size form (filled circles), which received the Imp1p processing but still remained outside the mitochondria, are plotted against incubation times in the left panel. Amounts of the total mature-size and intermediate-size forms (A, lanes 1–8, open squares), those of the protease-protected mature-size and intermediate-size forms (A, lanes 9–16, open circles), which were already localized in the IMS, and those of the protease-sensitive mature-size and intermediate-size forms (filled circles), which still remained outside of the mitochondria, are plotted against incubation times in the right panel. The amount of the fusion proteins added to the reaction is set to 100%. p, precursor; i, intermediate-size form; m, mature-size form.
Figure 3
Figure 3
The tight folding of the HBD promotes the Imp1p processing of the translocation intermediates of the pb2(220)-DHFR fusion proteins. (A) Radiolabeled pb2(220)WT-DHFR (WT), pb2(220)dc1-DHFR (dc1), and pb2(220)dc2-DHFR (dc2) were preincubated with 1 μM MTX/1 mM NADPH in import buffer for 15 min on ice and subsequently were incubated with yeast mitochondria at 25°C for indicated times. (B) Amounts of the intermediate-size forms and the mature-size forms. The amounts of the fusion proteins added to each reaction are set to 100%. p, precursor; i, intermediate-size form; m, mature-size form.
Figure 4
Figure 4
The tightly folded HBD in the IMS promotes the chase of the mature-size forms of the MTX-arrested pb2(220)-DHFR fusion proteins into the IMS when MTX is removed. (A) Radiolabeled pb2(220)WT-DHFR (WT), pb2(220)dc1-DHFR (dc1), and pb2(220)dc2-DHFR (dc2) were incubated with isolated yeast mitochondria at 25°C in the presence of 0.5 μM MTX. The mitochondria were reisolated by centrifugation, were washed once, and were incubated at 25°C for indicated times. The mitochondria were divided into halves and were incubated with (+ prot K, lanes 1–6) or without (− prot K, lanes 7–18) 100 μg/ml proteinase K for 30 min on ice. The mitochondria without proteinase K treatment were reisolated by centrifugation, and proteins recovered with the mitochondria (pellet) and those in the supernatant (sup) were analyzed by SDS/PAGE and radioimaging. p, precursor; i, intermediate-size form; m, mature-size form. (B) Amounts of the forms processed by Imp1p [the mature-size forms in “pellet” (A, lanes 7–12) and “sup” (A, lanes 13–18)] are plotted against incubation times in the left panel. Amounts of the imported proteins [the protease-protected mature-size forms (A, lanes 1–6)] are plotted against incubation times in the center panel; the amounts at time 0 are set to 0%. Amounts of the retrotranslocated proteins [the mature-size forms in “sup” (A, lanes 13–18)] are plotted against incubation times in the right panel; the amounts at time 0 are set to 0%. The amounts of the MTX-arrested fusion proteins associated with mitochondria after the first incubation are set to 100%. Squares, wild type (WT); triangles, dc1; circles, dc2.
Figure 5
Figure 5
Destabilization of the HBD in the IMS promotes the retrograde translocation of the kinetically arrested translocation intermediates of the pb2(260)-HBD fusion proteins. (A) pb2(260)-HBD fusion proteins. (B) Radiolabeled pb2(260)WT-HBD (WT), pb2(260)dc1-HBD (dc1), and pb2(260)dc2-HBD (dc2) were incubated with isolated yeast mitochondria in import buffer for 10 min at 25°C. The mitochondria were reisolated by centrifugation and were incubated at 25°C for indicated times. The mitochondria were divided into halves and were incubated with or without 100 μg/ml proteinase K for 30 min on ice. The mitochondria without proteinase K treatment were reisolated by centrifugation, and proteins recovered with the mitochondria (pellet) and those in the supernatant (sup) were analyzed by SDS/PAGE and radioimaging. Amounts of the forms processed by Imp1p (the mature-size forms in “pellet” and “sup”) are plotted against incubation times in the left panel. Amounts of the imported proteins (the protease-protected mature-size forms) are plotted against incubation times in the center panel; the amounts at time 0 are set to 0%. Amounts of the retrotranslocated proteins (the mature-size forms in “sup”) are plotted against incubation times in the right panel; the amounts at time 0 are set to 0%. The amounts of the kinetically arrested fusion proteins associated with mitochondria after the first incubation are set to 100%. Squares, wild type (WT); triangles, dc1; circles, dc2.
Figure 6
Figure 6
The stability of the HBD in the IMS does not affect the chase of the intermediate-size forms of the MTx-arrested pb2(220)-DHFR fusion proteins into the IMS when MTX is removed. (A) pb2AA(220)-DHFR fusion proteins. (B) Generation of the translocation intermediates of radiolabeled pb2AA(220)WT-DHFR, pb2AA(220)dc1-DHFR, and pb2AA(220)dc2-DHFR at the outer membrane and the subsequent chase reactions were performed as described for Fig. 4. Amounts of the imported proteins (the protease-protected intermediate-size forms) are plotted against incubation times. Squares, AA; triangles, AA/dc1; circles, AA/dc2.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Hanein D, Matlack K E S, Jungnickel B, Plath K, Kalies K-U, Miller K R, Rapoport T A, Akey C W. Cell. 1996;87:721–732. - PubMed
    1. Künkele K-P, Heins S, Dembowski M, Nargang F E, Benz R, Thieffry M, Walz J, Lill R, Nussberger S, Neupert W. Cell. 1998;93:1009–1019. - PubMed
    1. Meyer T H, Ménétret J-F, Breitling R, Miller K R, Akey C W, Rapoport T A. J Mol Biol. 1999;285:1789–1800. - PubMed
    1. Hartl F-U, Neupert W. Science. 1990;247:930–938. - PubMed
    1. Glick B S, Beasley E M, Schatz G. Trends Biochem Sci. 1992;17:453–459. - PubMed

Publication types

LinkOut - more resources