Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane

Abstract

Little is known about how the photosynthetic machinery is arranged in time and space during the biogenesis of thylakoid membranes. Using in situ cryo-electron tomography to image the three-dimensional architecture of the cyanobacterium Synechocystis, we observed that the tips of multiple thylakoids merge to form a substructure called the ‘convergence membrane’. This high-curvature membrane comes into close contact with the plasma membrane at discrete sites. We generated subtomogram averages of 70S ribosomes and array-forming phycobilisomes, then mapped these structures onto the native membrane architecture as markers for protein synthesis and photosynthesis, respectively. This molecular localization identified two distinct biogenic regions in the thylakoid network: thylakoids facing the cytosolic interior of the cell that were associated with both marker complexes, and convergence membranes that were decorated by ribosomes but not phycobilisomes. We propose that the convergence membranes perform a specialized biogenic function, coupling the synthesis of thylakoid proteins with the integration of cofactors from the plasma membrane and the periplasmic space.

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Fig. 1: In situ cryo-ET of wild-type Synechocystis cells showing the heterogeneity of membrane architecture at thylakoid convergence zones.
Fig. 2: Thylapse gallery.
Fig. 3: Native in situ structure of the Synechocystis phycobilisome array.
Fig. 4: Phycobilisome arrays are bound to PSII.
Fig. 5: Native in situ structures of Synechocystis cytosolic ribosomes.
Fig. 6: Concentrations of phycobilisomes and membrane-associated ribosomes at different membrane regions of Synechocystis.

Data availability

Subtomogram averages have been deposited in the Electron Microscopy Data Bank (EMD-4599 to EMD-4602), along with the tomograms shown in Fig. 1a,d (EMD-4603 and EMD-4604). Additional data that support the findings of this study are available from the corresponding authors upon request.

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Acknowledgements

The authors thank W. Baumeister for enabling this project by providing support and instrumentation, D. Tegunov for creating the membranogram software, R. Danev for help with the phase plate, as well as S. Heinz and G. Weiss for discussion. This work was supported by grants awarded to B.D.E. (EN 1194/1-1) and J.N. (NI 390/9-2) by the Deutsche Forschungsgemeinschaft in the context of the Research Unit FOR2092. Additional funding was provided by the Max Planck Society and by LMU Munich’s Institutional Strategy “LMU Excellent” within the framework of the German Excellence Initiative.

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Contributions

A.R. and M.S. cultured and froze cells. M.S. performed the FIB milling. A.R., M.S., S.A. and B.D.E. acquired the tomograms. A.R. performed all the analyses in the paper, with assistance from the following people: W.W. guided subtomogram averaging using STOPGAP, producing the phycobilisome structure shown in Fig. 3; S.A. guided subtomogram averaging using PyTom, producing the phycobilisome structure shown in Fig. 4; S.P. helped with the averaging and interpretation of the ribosome structure shown in Fig. 5; and F.B. provided additional computational guidance. J.M.P. enabled instrumentation and provided funding. J.N. and B.D.E. provided funding and conceived and supervised the project. A.R., J.N. and B.D.E. wrote the paper, with input from all authors.

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Correspondence to Jörg Nickelsen or Benjamin D. Engel.

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Journal peer review information Nature Plants thanks Matthew Johnson and other anonymous reviewers for their contribution to the peer review of this work.

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Supplementary Information

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Video Legends.

Reporting Summary

Supplementary Video 1

In situ cryo-ET of the Synechocystis thylakoid convergence zone (overview 1). Accompanies Fig. 1a and b. The movie slices back and forth through the tomographic volume from Fig. 1a, then reveals the 3D segmentation and mapped in macromolecules shown in Fig. 1b. First, the cytosolic ribosomes (grey) are hidden to show the biogenic regions of the thylakoid network (green), decorated with membrane-associated ribosomes (pink). Photosynthetic regions are decorated with phycobilisomes (yellow). Later, the outer membrane (light blue), plasma membrane (dark blue) and associated macromolecules are hidden for a close-up view of the convergence membrane and thylapse contact.

Supplementary Video 2

In situ cryo-ET of the Synechocystis thylakoid convergence zone (overview 2). Accompanies Fig. 1e and f. The movie slices back and forth through the tomographic volume from Fig. 1e, then reveals the 3D segmentation and mapped in macromolecules shown in Fig. 1f. First, the cytosolic ribosomes (grey) are hidden to show the biogenic regions of the thylakoid network (green), decorated with membrane-associated ribosomes (pink). Photosynthetic regions are decorated with phycobilisomes (yellow). Later, the outer membrane (light blue), plasma membrane (dark blue) and associated macromolecules are hidden for a close-up view of the convergence membrane and thylapse contact.

Supplementary Video 3

Close-up views of convergence zone and thylapse architecture. Accompanies Fig. 2. The movie slices back and forth through tomographic volumes corresponding to Fig. 2a,d,g–i,k and l. An extra volume is shown from the Fig. 2k tomogram in a region not displayed in the figure that contains an exemplary convergence membrane, but not a thylapse. For each volume, it is indicated whether defocus or the Volta phase plate was used to generate contrast.

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Rast, A., Schaffer, M., Albert, S. et al. Biogenic regions of cyanobacterial thylakoids form contact sites with the plasma membrane. Nat. Plants 5, 436–446 (2019). https://doi.org/10.1038/s41477-019-0399-7

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