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Reconstitution of the tubular endoplasmic reticulum network with purified components

Nature volume 543, pages 257–260 (2017)Cite this article

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Abstract

Organelles display characteristic morphologies that are intimately tied to their cellular function, but how organelles are shaped is poorly understood. The endoplasmic reticulum is particularly intriguing, as it comprises morphologically distinct domains, including a dynamic network of interconnected membrane tubules. Several membrane proteins have been implicated in network formation1,2,3,4,5, but how exactly they mediate network formation and whether they are all required are unclear. Here we reconstitute a dynamic tubular membrane network with purified endoplasmic reticulum proteins. Proteoliposomes containing the membrane-fusing GTPase Sey1p (refs 6, 7) and the curvature-stabilizing protein Yop1p (refs 8, 9) from Saccharomyces cerevisiae form a tubular network upon addition of GTP. The tubules rapidly fragment when GTP hydrolysis of Sey1p is inhibited, indicating that network maintenance requires continuous membrane fusion and that Yop1p favours the generation of highly curved membrane structures. Sey1p also forms networks with other curvature-stabilizing proteins, including reticulon8 and receptor expression-enhancing proteins (REEPs)10 from different species. Atlastin, the vertebrate orthologue of Sey1p6,11, forms a GTP-hydrolysis-dependent network on its own, serving as both a fusion and curvature-stabilizing protein. Our results show that organelle shape can be generated by a surprisingly small set of proteins and represents an energy-dependent steady state between formation and disassembly.

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Figure 1: Co-reconstituted Sey1p and Yop1p form GTP-dependent tubular networks.
Figure 2: Network maintenance requires continuous GTP hydrolysis by Sey1p.
Figure 3: Visualization of reconstituted networks with negative-stain electron microscopy.
Figure 4: Networks formed with different membrane-fusing and curvature-stabilizing proteins.

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Acknowledgements

We thank H. Tukachinsky for help with protein purifications, M. Kozlov for discussions, A. Daga for material, the Nikon Imaging Center at Harvard Medical School, the Harvard Medical School electron microscopy facility, and the ICCB-Longwood Screening Facility for help. We thank M. Kozlov, J. Hu, and H. Tukachinsky for reading the manuscript. R.E.P. is supported by National Institutes of Health/National Institute of General Medical Sciences T32 GM008313 training grant. S.W. is supported by a fellowship from the Charles King Trust. T.A.R. is a Howard Hughes Medical Institute Investigator.

Author information

Author notes

  1. Tina Y. Liu

    Present address: † Present address: 731 Stanley Hall, MS 3220, University of California, Berkeley, California 94720-3220, USA.,

  2. Robert E. Powers and Songyu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Robert E. Powers, Songyu Wang, Tina Y. Liu & Tom A. Rapoport

  2. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, 02115, Massachusetts, USA

    Robert E. Powers, Songyu Wang, Tina Y. Liu & Tom A. Rapoport

Authors
  1. Robert E. Powers
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  2. Songyu Wang
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Contributions

R.E.P. and S.W. performed all experiments. Initial tests of Sey1p and Yop1p co-reconstitution were performed by T.Y.L. R.E.P., S.W., and T.A.R. designed the experiments and wrote the paper. T.A.R. supervised the project.

Corresponding author

Correspondence to Tom A. Rapoport.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Purity of ER-shaping proteins used in reconstitution experiments.

The indicated proteins were purified and subjected to SDS–PAGE and Coomassie blue staining.

Extended Data Figure 2 Orientation of proteins after reconstitution into liposomes.

a, D. melanogaster ATL was reconstituted into rhodamine-PE-labelled liposomes at a protein:lipid ratio of 1:1,000. The vesicles were incubated with decreasing concentrations of trypsin in the absence (left) or presence (right) of 0.2% Triton X-100 for 30 min at room temperature. Samples were analysed by SDS–PAGE and Coomassie blue staining. b, As in a, but with S. cerevisiae Sey1p at a protein:lipid ratio of 1:500. c, As in a, but with S. cerevisiae Yop1p at a protein:lipid ratio of 1:100. d, As in a, but with Sey1p and Yop1p at protein:lipid ratios of 1:500 and 1:100, respectively. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 3 Flotation of proteoliposomes generated with Sey1p and curvature-stabilizing proteins.

a, S. cerevisiae Sey1p and Yop1p were co-reconstituted into rhodamine-PE-labelled liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. The samples were centrifuged in a Nycodenz gradient, and fractions (F1–F6) were collected from the top and analysed by SDS–PAGE and Coomassie blue staining. b, As in a, but with proteins only in the presence of 0.03% DDM. c, As in a, but with proteoliposomes containing S. cerevisiae Sey1p and Rtn1p at protein:lipid ratios of 1:500 and 1:50, respectively. d, As in c, but with proteins only in the presence of 0.03% DDM. e, As in a, but with proteoliposomes containing Sey1p and D. melanogaster Rtn1p at protein:lipid ratios of 1:500 and 1:50, respectively. f, As in e, but with proteins only in the presence of 0.03% DDM. g, As in a, but with proteoliposomes containing Sey1p and D. melanogaster CG8331 at protein:lipid ratios of 1:500 and 1:50, respectively. h, As in g, but with proteins only in the presence of 0.03% DDM. i, As in a, but with proteoliposomes containing Sey1p and X. laevis REEP5 at protein:lipid ratios of 1:500 and 1:200, respectively. j, As in i, but with proteins only in the presence of 0.03% DDM. k, As in a, but with proteoliposomes containing Sey1p and D. melanogaster ATL(K51A) at protein:lipid ratios of 1:500 and 1:100, respectively. l, As in k, but with proteins only in the presence of 0.03% DDM. m, As in a, but with proteoliposomes containing D. melanogaster ATL at protein:lipid ratios of 1:500 or 1:1,000. n, As in a, but with proteoliposomes containing S. cerevisiae Sey1p at protein:lipid ratios of 1:500 or 1:1,000. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 4 Fusion activity of Sey1p-containing proteoliposomes.

a, Proteoliposomes were generated with either S. cerevisiae Sey1p alone (protein:lipid ratio of 1:500) or with Sey1p and S. cerevisiae Yop1p (protein:lipid ratios of 1:500 and 1:100, respectively). Donor vesicles contained NBD-PE and rhodamine-PE. After addition of 1 mM GTP, fusion with unlabelled acceptor vesicles was measured by dequenching of the NBD fluorescence. Controls were performed in the absence of GTP. b, As in a, but with Sey1p and Yop1p at protein:lipid ratios of 1:1,000 and 1:200, respectively. Each curve corresponds to the mean of three biological replicates.

Extended Data Figure 5 Reconstituted networks display heterogeneity.

S. cerevisiae Sey1p and Yop1p were incorporated into rhodamine-PE-labelled liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. The proteoliposomes were incubated with 2 mM GTP, spotted on a cover slip, and imaged with a fluorescence microscope. Shown are different areas from the same coverslip. Note that the networks differ with respect to the density of three-way junctions and length of tubules. Scale bars, 20 μm.

Extended Data Figure 6 Control experiments for network formation.

a, S. cerevisiae Sey1p and Yop1p were co-reconstituted into rhodamine-PE-labelled liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. The proteoliposomes were incubated with either 2 mM GTP or GTP-γS and visualized by fluorescence microscopy. b, Proteoliposomes containing only S. cerevisiae Sey1p at a protein:lipid ratio of 1:500 were incubated in the absence of GTP. The same sample is shown incubated with GTP in Fig. 1d. c, As in b, but with proteoliposomes containing only S. cerevisiae Yop1p at a protein:lipid ratio of 1:35. The same sample is shown incubated with GTP in Fig. 1e. Scale bars, 20 μm.

Extended Data Figure 7 Network formation with different concentrations of Yop1p and Sey1p.

a, S. cerevisiae Sey1p was co-reconstituted with S. cerevisiae Yop1p into rhodamine-PE-labelled liposomes at protein:lipid ratios of 1:500 and 1:200, respectively (instead of the usual 1:500 and 1:35 ratios). The proteoliposomes were incubated with or without 2 mM GTP and visualized by fluorescence microscopy. b, As in a, but with protein:lipid ratios of 1:1,000 and 1:200, respectively. Scale bars, 20 μm.

Extended Data Figure 8 Tubular network formation with different lipid compositions.

a, S. cerevisiae Sey1p and Yop1p were co-reconstituted into rhodamine-PE-labelled liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. The liposomes were generated with a polar lipid extract from E. coli. The proteoliposomes were incubated with or without 2 mM GTP and visualized with a fluorescence microscope. b, As in a, but with liposomes generated with a polar lipid extract from S. cerevisiae. Scale bars, 20 μm.

Extended Data Figure 9 Sey1p-containing networks visualized with negative-stain electron microscopy.

a, S. cerevisiae Sey1p and Yop1p were co-reconstituted into liposomes at protein:lipid ratios of 1:1,000 and 1:200, respectively. The samples were incubated with 1 mM GTP and visualized by electron microscopy after staining with uranyl acetate. The boxed area of this network is shown enlarged in Fig. 3a. b, As in a, but with Sey1p and Yop1p at protein:lipid ratios of 1:7,500 and 1:200, respectively. Black arrowheads indicate Sey1p molecules. c, As in b, showing another area (left). Boxed area is shown enlarged (right) with black arrowheads indicating Sey1p molecules and the dotted black line traces approximate plane of the lipid bilayer. d, As in a, but with Sey1p and D. melanogaster Rtnl1 at protein:lipid ratios of 1:500 and 1:50, respectively, in the absence or presence of 1 mM GTP. e, As in a, but with D. melanogaster ATL D. melanogaster Rtnl1 at protein:lipid ratios of 1:2,500 and 1:200, respectively, in the absence or presence of 1 mM GTP. Scare bars, 100 nm.

Extended Data Figure 10 Network formation with different membrane-fusing and curvature-stabilizing proteins requires GTP hydrolysis.

The samples shown in Fig. 4 were incubated without GTP. Scale bars, 20 μm.

Supplementary information

Supplementary Figure 1

Uncropped source gels for gels depicted in Extended Figures 2 and 3. (PDF 622 kb)

Dynamics of a reconstituted network

S. cerevisiae Sey1p and Yop1p were co-reconstituted into rhodamine-PE labeled liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. The proteoliposomes were incubated in the presence of 2 mM GTP and visualized by fluorescence microscopy. The sample was imaged every 0.5 sec for 15 sec. The video is shown at 2 frames per sec. White arrows indicate sliding or fusing junctions that are shown in a magnified view in Fig. 1c. Cyan arrows indicate other sliding or fusing junctions. Frames from this video are shown in Fig 1c. Scale bars = 20 μm. (MOV 3183 kb)

Fragmentation of a reconstituted network after addition of GTPγS

S. cerevisiae Sey1p and Alexa647-labeled Yop1p were co-reconstituted into rhodamine-PE containing liposomes at protein:lipid ratios of 1:500 and 1:35, respectively. Network was formed by incubating proteoliposomes with 2 mM GTP. After addition of 1 mM GTPγS, the samples were analyzed by fluorescence microscopy. The sample was imaged every sec for 30 sec. The video is shown at 2 frames per sec and displays the Alexa647-labeled Yop1p. Cyan arrows indicate points of fragmentation. Frames from this video are shown in Fig 2b. Scale bars = 20 μm. (MOV 14834 kb)

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Powers, R., Wang, S., Liu, T. et al. Reconstitution of the tubular endoplasmic reticulum network with purified components. Nature 543, 257–260 (2017). https://doi.org/10.1038/nature21387

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