Abstract
The conserved oligomeric Golgi (COG) complex orchestrates vesicular trafficking to and within the Golgi apparatus. Here, we use negative-stain electron microscopy to elucidate the architecture of the hetero-octameric COG complex from Saccharomyces cerevisiae. Intact COG has an intricate shape, with four (or possibly five) flexible legs, that differs strikingly from that of the exocyst complex and appears to be well suited for vesicle capture and fusion.
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Acknowledgements
This work was supported by NIH grants R01 GM071574 (F.M.H.) and P01 GM062580 (T.W.).
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All authors designed experiments and analyzed data. J.Y.H., H.-T.C., D.U., and C.K.Y. performed the experiments. J.Y.H., H.-T.C., T.W., and F.M.H. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Purification and negative-stain EM of K. lactis Cog5–8.
(a) The purified recombinant K. lactis Cog5-8 complex, visualized by SDS-PAGE and Coomassie Blue staining, is shown together with a representative image field and ten representative class averages. (b) Complete gallery of K. lactis Cog5-8 class averages (side length of each panel = 38 nm). The 129 class averages, which account for 4,958 particles, were obtained by subjecting 8,825 particles to 14 generations of ISAC.
Supplementary Figure 2 Negative-stain EM of S. cerevisiae Cog1–8.
Complete gallery of S. cerevisiae Cog1-8 class averages (side length of each panel = 63 nm). The 278 class averages, which account for 5,182 particles, were obtained by subjecting 31,872 particles to 18 generations of ISAC.
Supplementary Figure 3 Localizing individual subunits within the K. lactis Cog5–8 complex.
(a) Purified K. lactis Cog5-8 complexes containing single GFP tags, visualized by SDS-PAGE and Coomassie Blue staining. GFP-Cog6ΔN denotes N-terminally truncated Cog6 (residues 147-779). (b) Complete galleries of GFP-tagged K. lactis Cog5-8 class averages (side length of each panel = 38 nm). Only those complexes in which the GFP tag was visible in class averages are included; the remaining complexes were indistinguishable from the untagged complexes shown in Supplementary Fig. 1b.
Supplementary Figure 4 Dimensions of S. cerevisiae Cog1–8.
The indicated lengths (mean ± s.d.; n=5) were derived from class averages using ImageJ (https://imagej.nih.gov/ij/).
Supplementary Figure 5 Negative-stain EM of bovine COG.
Shown are four additional image fields of the bovine COG complex (see also Fig. 2e). The particles were too heterogeneous to allow successful class averaging.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Table 1 (PDF 1662 kb)
Supplementary Data Set 1
Uncropped gel for Fig. 1 (PDF 489 kb)
Yeast COG appears to contain flexible hinges
Negative-stain EM of S. cerevisiae Cog1–8 yields class averages in which the legs of the complex adopt various orientations. (MOV 163 kb)
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Ha, J., Chou, HT., Ungar, D. et al. Molecular architecture of the complete COG tethering complex. Nat Struct Mol Biol 23, 758–760 (2016). https://doi.org/10.1038/nsmb.3263
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DOI: https://doi.org/10.1038/nsmb.3263
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