Abstract
We show here that the Saccharomyces cerevisiae GARP complex and the Cog1–4 subcomplex of the COG complex, both members of the complexes associated with tethering containing helical rods (CATCHR) family of multisubunit tethering complexes, share the same subunit organization. We also show that HOPS, a tethering complex acting in the endolysosomal pathway, shares a similar architecture, thus suggesting that multisubunit tethering complexes use related structural frameworks.
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Acknowledgements
We thank D. Finley and S. Elsasser (Harvard Medical School) for providing vectors and advice on generating the yeast strains used for N-terminal GFP tagging. This work was supported by NIH grant P01 GM062580 (to T.W.). K.M.R. is supported by GM80616.
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T.W. and H.-T.C. designed the experiments and analyzed the data. H.-T.C. performed all the experiments. D.D. assisted with preparing GFP-labeled yeast strains, and M.G.C. assisted with EM imaging and data processing. T.W., K.M.R., and H.-T.C. wrote the manuscript.
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Integrated supplementary information
Supplementary Figure 1 TAP purification and negative-stain EM analysis of untagged yeast GARP complex and GARP complex with C-terminally GFP-tagged Vps51.
(a) The silver-stained SDS-PAGE gel shows that the peak fraction from the TAP purification contains all four subunits of the GARP complex. The TAP-tagged Vps54 subunit used for purification retains the calmodulin-binding protein (CBP). MW, molecular weight standards in kDa. (b) A representative EM image of negatively stained GARP complex. Some of the Y-shaped complexes are circled. The scale bar is 50 nm. (c) The 231 averages of negatively stained GARP complex obtained with the ISAC procedure, revealing the flexibility of the legs in the Y-shaped complex. The asterisks indicate the averages shown in Figure 1a. The side length of the individual panels is 49.2 nm. (d) SDS-PAGE gel of the purified GARP complex with GFP-tagged Vps51, which was silver stained (left lane) and Western blotted with an anti-GFP antibody (right lane). The TAP-tagged Vps54 subunit used for purification retains the calmodulin-binding protein (CBP). (e) Representative negative-stain EM image. Some of the GFP-tagged GARP complexes are circled. The scale bar is 50 nm. (f) The 50 averages obtained with K-means classification. Only few of the averages resolve all three legs of the GARP complex and the extra density for the GFP. The asterisks indicate the averages shown in Figure 1b. The side length of the individual panels is 49.2 nm.
Supplementary Figure 2 Purification of double-GFP-tagged GARP complexes.
(a) SDS-PAGE gels of purified, double-GFP-tagged GARP complexes that were silver stained (left lanes) and Western blotted with an anti-GFP antibody (right lanes). All GARP complexes contain C-terminally GFP-tagged Vps51 as well as a GFP on the N or C terminus of one of the other GARP subunits. The positions of the two GFP tags are denoted above the gels. The TAP-tagged subunits used for purification retain the calmodulin-binding protein (CBP). (b) Top panel: SDS-PAGE gel of purified GARP complex containing N-terminally GFP-tagged Vps51 and C-terminally GFP-tagged Vps54 that was silver stained (left lane) and Western blotted with an anti-GFP antibody (right lane). The TAP-tagged Vps52 subunit used for purification retains the calmodulin-binding protein (CBP). Bottom panel: Representative negative-stain EM image. Some of the double-GFP-tagged GARP complexes are circled. EM images of the other double-GFP-tagged complexes shown in (a) were of similar quality (not shown). The scale bar is 50 nm.
Supplementary Figure 3 Negative-stain EM images of double-GFP-tagged GARP complexes and of GARP complex lacking Vps51.
(a - f) Raw particle images (top panels) and schematic drawings (bottom panels) of GARP complexes with a GFP at the N terminus (a) or C terminus (b) of Vps52, at the N terminus (c) or C terminus (d) of Vps53, and at the N terminus (e) or C terminus (f) of Vps54. A C-terminal GFP on Vps51 was used to mark leg C. The side length of the individual panels is 49.2 nm. (g) Raw particle images (top panels) and schematic drawings (bottom panels) of GARP complexes with a GFP at the N terminus of Vps51. A C-terminal GFP on Vps54 was used to mark leg C. The side length of the individual panels is 49.2 nm. (h) Left panel: Silver-stained SDS-PAGE gel of GARP complex purified from a ΔVps51 strain. Vps54 carried a C-terminal GFP and Vps52 was TAP-tagged. Right: Representative negative-stain EM image showing only partially assembled GARP complexes. The scale bar is 50 nm.
Supplementary Figure 4 Comparison of the C termini of Vps53 and Cog4 and purification of the HOPS complex.
(a, b) Crystal structures show that the C-terminal domains of the corresponding subunits, Vps53 of the S. cerevisiae GARP complex (PDB ID: 3NS4) (Vasan, N. et al., Proc. Natl. Acad. Sci. USA 107, 14176–14181, 2010) and Cog4 of the human COG complex (PDB ID: 3HR0) (Richardson, B.C. et al., Proc. Natl. Acad. Sci. USA 106, 13329–13334, 2009) have a similar arrangement of α-helices. Distinct helical bundles are colored in red, green, blue and purple. The enlarged views show residues Gln695 of Vps53 and Arg729 of Cog4, which are located in conserved patches and cause diseases when mutated (residue Gln624 in yeast Vps53 corresponds to Gln695 in the human homolog). (c - e) TAP purification and EM analysis of the yeast HOPS complex. (c) Silver-stained SDS-PAGE gel of the HOPS complex purified using a TAP tag on Vps41, which retains the calmodulin-binding protein (CBP). MW, molecular weight standards in kDa. The bracket indicates the region of the gel that was analyzed by mass spectrometry. (d) Representative negative-stain EM image in which some HOPS complexes are circled. The scale bar is 100 nm. (e) Effect of chemical cross-linking on the appearance of the HOPS complex. The negative-stain EM image of cross-linked HOPS complex shows that the complexes are more compact and lack the legs seen in negatively stained samples prepared without cross-linking. Some complexes are circled. The scale bars are 100 nm.
Supplementary Figure 5 EM analysis of the HOPS complex and its proposed subunit organization.
(a) The 238 class averages of negatively stained HOPS complex obtained with the ISAC procedure. The numbers indicate the averages shown in Figure 2a. The side length of the individual panels is 67.2 nm. (b) Schematic representation of the subunit organization of the HOPS complex published before (Bröcker, C. et al., Proc. Natl. Acad. Sci. USA 109, 1991–1996, 2012). Vps39 (green) and Vps41 (blue) are substituted in the CORVET complex by Vps3 and Vps8, respectively. (c) An ISAC average of negatively stained HOPS complex showing the globular head, consisting of a bipartite larger domain and a globular smaller domain, I and II, respectively, and three legs, A to C. (d) Crystal structure of Vps33 (red) in complex with the C-terminal domain of Vps16 (residues 505-834) (blue) (PDB ID: 4KMO) (Baker, R.W. et al., PLoS ONE 8, e67409, 2013). The crystal structure of the N-terminal β-propeller of Vps18 (yellow) (PDB ID: 4UUY) (Behrmann, H. et al., J. Biol. Chem. 289, 33503–33512, 2014) is shown to represent the N-terminal domain of Vps16. (e) A class average of negatively stained HOPS complex obtained by K-means classification. While the legs are largely averaged out, the average shows more structural features for the head domain. The black lines indicate the correspondence between the atomic model in panel (d) and domain I of the class average. (f) The 50 class averages of negatively stained HOPS complex obtained by K-means classification. The white box indicates the region shown in panel (e). The side length of the individual panels is 67.2 nm. (g) Schematic drawing of the subunit organization of the HOPS complex deduced before (Ostrowicz, C.W. et al., Traffic 11, 1334–1346, 2010). Vps39 (green) and Vps41 (blue) are substituted in the CORVET complex by Vps3 and Vps8, respectively. The gray level of the remaining subunits represents the number of other subunits it interacts with: Vps11 interacts with four other subunits, Vps16 and Vps18 each interact with three other subunits, and Vps33 interacts only with one other subunit. (h) The ISAC average shown in panel (c) with the structural features labeled with the names of the subunits proposed to constitute them.
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Chou, HT., Dukovski, D., Chambers, M. et al. CATCHR, HOPS and CORVET tethering complexes share a similar architecture. Nat Struct Mol Biol 23, 761–763 (2016). https://doi.org/10.1038/nsmb.3264
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DOI: https://doi.org/10.1038/nsmb.3264
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