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Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins


Some proteins require completion of folding before translocation across a membrane into another cellular compartment. Yet the permeability barrier of the membrane should not be compromised and mechanisms have remained mostly elusive. Here, we present the structure of Saccharomyces cerevisiae Bcs1, an AAA-ATPase of the inner mitochondrial membrane. Bcs1 facilitates the translocation of the Rieske protein, Rip1, which requires folding and incorporation of a 2Fe–2S cluster before translocation and subsequent integration into the bc1 complex. Surprisingly, Bcs1 assembles into exclusively heptameric homo-oligomers, with each protomer consisting of an amphipathic transmembrane helix, a middle domain and an ATPase domain. Together they form two aqueous vestibules, the first being accessible from the mitochondrial matrix and the second positioned in the inner membrane, with both separated by the seal-forming middle domain. On the basis of this unique architecture, we propose an airlock-like translocation mechanism for folded Rip1.

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Fig. 1: Bcs1 structure and domain organization in the ADP state.
Fig. 2: Bcs1 vestibules and protomer interactions.
Fig. 3: The IM vestibule and TMH surface hydrophobicity.
Fig. 4: Overview of the Bcs1 ADP and apo states.
Fig. 5: Conformational dynamics of Bcs1 between ADP and apo states.
Fig. 6: Model for translocation of folded Rip1 protein by Bcs1.

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Data availability

The cryo-EM density maps and corresponding atomic models reported in this paper have been deposited in the Electron Microscopy Data Bank and Protein Data Bank with the accession codes EMD-10192, EMD-10193 and EMD-10194, as well as PDB 6SH3, 6SH4 and 6SH5, for the Bcs1-ADP, Bcs1-Apo1 and Bcs1-Apo2 states, respectively.


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We thank S. Rieder, C. Ungewickell and S. Esser for technical assistance and help with experiments and M. Ameismeier for support with electron microscopy. This work was supported by funding from the Deutsche Forschungsgemeinschaft (DFG) through the GRK1721 to R.B., a DFG fellowship through the QBM (Quantitative Biosciences Munich) graduate school to L.K. and the DFG grant WA3802/1-1 and the LMUexcellent program of the Bundesexzellenzinitiative to N.W. and W.N.

Author information

Authors and Affiliations



R.B. and W.N. designed the study and L.K., N.W., T.B. and R.B. wrote the manuscript. N.W. purified and characterized Bcs1 complexes, L.K. prepared cryo-EM samples and, together with O.B., collected data. L.K. processed cryo-EM maps, built molecular models and, together with T.B. and R.B., interpreted the structures.

Corresponding author

Correspondence to Roland Beckmann.

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The authors declare no competing interests.

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Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 SDS-PAGE gel of purified Bcs1.

The concentrated Bcs1-His eluate (see Materials and Methods) was loaded on a 4-10% gradient gel and bands were analyzed by mass spectrometry. This mild and fast one-step affinity purification yields Bcs1 assemblies active in nucleotide and substrate binding7; remaining background proteins represent abundant mitochondrial proteins that unspecifically interact with Ni-NTA resin.

Extended Data Fig. 2 Cryo-EM data processing of the Bcs1 complex with and without ADP.

(a) Representative original micrograph and 2D class averages for the ADP (left) and the Apo (right) datasets. (b) “Gold standard” Fourier-Shell correlation (FSC) curves when applying C1 and C7 symmetry during refinement. The final resolutions were 3.4 Å (C7) and 4.0 Å (C1). (c) Final C1 and C7 symmetrized maps filtered and colored according to local resolution as calculated with RELION-3.

Extended Data Fig. 3 3D classification scheme for the Bcs1 apo data set.

The classification regiment is described in detail in the Methods section. In brief, after import and CTF-estimation, particles were first picked using the structure of the Vps4 AAA-ATPase hexamer as a template (EMD-8588)37. Particles were 2D classified and an ab initio model was obtained using cryoSPARC v.2. From the refined map, a better reference for RELION autopicking was obtained and resulting particles were again 2D classified. Good classes were subjected to 3D classification yielding one stable feature-containing class that could be sub-classified further. Two stable classes were then 3D refined using either C1 or C7 symmetry yielding the final Bcs1 Apo1 and Bcs1 Apo2 maps. K: number of classes.

Extended Data Fig. 4 3D classification scheme for the Bcs1 ADP dataset.

The classification regiment is described in detail in the Methods section. In brief, autopicking in RELION-3 (Laplacian of Gaussian (LoG) method)38 was performed using the previously obtained structure of Bcs1 Apo1 as a reference. 2D classification and 3D refinement were performed and auto-picking repeated using the refined map to optimize the quality and yield of good Bcs1 complex particles. After a second round of 2D classification and refinement, 3D classification yielded in one class that could be refined including per particle CTF correction and beam tilt correction in RELION-3 using C1 and C7 symmetry to a final resolution of and 3.4 Å and 4.0 Å, respectively. K: number of classes.

Extended Data Fig. 5 Comparison of two membrane bound mitochondrial AAA proteins Yme1 and Bcs1.

Shown are two protomers of both Yme1 (a) (PDB: 6AZ0)20 and Bcs1 either in the ADP state (b) or the Apo1 state (c). The pore loops and preceding beta strands are shown in orange (PL1, β2) and blue (PL2, β3). The six AAA domains of Yme1 form a spiral staircase configuration. Displayed here are the ADP-bound (purple) and Apo (cyan) protomers, which constitute the bottom and top end of this spiral. The two pore loops of every AAA domain protrude towards the central axis of this complex, where they can interact with the peptide backbone of the substrate (red) via the conserved tyrosine residues. In this configuration, the beta strands β2 and β3, which precede the two pore loops point towards the central axis. Conversely, in Bcs1, these loops and the corresponding preceding beta strands do not point towards the opening of the ring of AAA domains, but rather point away from the ring plane in both displayed conformations. In the ADP state these loops are oriented approximately parallel to the central axis, in the apo states they are slightly tilted towards the central axis.

Supplementary information

Reporting Summary

Supplementary Video 1

Overview of Bcs1 and transitions between the Bcs1 ADP and apo states.

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Kater, L., Wagener, N., Berninghausen, O. et al. Structure of the Bcs1 AAA-ATPase suggests an airlock-like translocation mechanism for folded proteins. Nat Struct Mol Biol 27, 142–149 (2020).

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