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. 2021 Sep 14;12:5421. doi: 10.1038/s41467-021-25724-y

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Schematic representation of the MAD2L2 protein with residues mutated to abolish dimerization. b Immunoblot of TRF2ts MEFs transduced with indicated shRNAs, complemented with shRNA-resistant expression constructs (EV, empty vector control; WT, full-length wild-type MAD2L2; 1xMut and 3xMut as indicated in a, all with single Flag-tag). HSP90 serves as loading control. c Immunoblot of 293T cells transfected with indicated epitope-tagged constructs followed by Flag immunoprecipitation. V5-SHLD2 is detected with V5-antibody, GFP-SHLD3 with GFP-antibody and Flag-MAD2L2 (Fl-MAD2L2) with MAD2L2-antibody. Asterisk denotes immunoglobulin light-chain. Representative of three independent experiments. d SHLD2^1-95 induces dimerization of MAD2L2-SHLD3 complex. Coomassie-stained SDS-PAGE gels of SEC-profiles of SHLD3-MAD2L2 complex (WT: light green, 1xMut: dark green) and SHLD3-SHLD2^1-95-MAD2L2 complex (WT: red, 1xMut: orange). Shift in elution-profile indicates an apparent increase of molecular weight of ~54 to ~90 kDa upon addition of SHLD2^1-95. Molecular weight of MAD2L2 is 25 kDa, of SHLD3 is 29 kDa and of SHLD2^1-95 is 11 kDa. e Time course of shieldin assembly between components MAD2L2 and SHLD3 shows that assembly of SHLD3-MAD2L2 complex is slow, but accelerated by MAD2L2 dimerization. Coomassie-stained SDS-PAGE gels show a time course of pulldowns of MBP-SHLD3 (bait) and MAD2L2-variants (prey). The SHLD3-fragment used here contains only the second RBM, which is captured by the seatbelt of MAD2L2²⁹. A single-point mutation at R124 (1xMut) hinders MAD2L2 dimerization and subsequently lowers binding kinetics. Triple mutation of residues K44, R124, and A135 (3xMut) in the dimerization interface of MAD2L2 has the same effect as 1xMut on binding kinetics. Inducing dimerization by creating GST-fusion partially rescues the assembly kinetics. Representative of n = 4 (MAD2L2^WT/MAD2L2^1xMut) or n = 2 (MAD2L2^3xMut/GST-MAD2L2^1xMut). f Graphical representation of data in e. Red curve represents binding kinetics between MAD2L2^WT and SHLD3^28-83 peptide. Light blue and green curves represent the reduction in binding kinetics of MAD2L2^1xMut and MAD2L2^3xMut with SHLD3^28-83. Dark blue curve represents partial restoration of binding kinetics between MAD2L2^1xMut and SHLD3^28-83 induced by dimerization of the GST-tag. Graphs represent mean ± s.e.m, n = 4 for MAD2L2^WT/MAD2L2^1xMut, n = 2 for MAD2L2^3xMut/GST-MAD2L2^1xMut. Source data are provided as a Source Data file.