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. 2019 Dec 20;10:5807. doi: 10.1038/s41467-019-13864-1

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© The Author(s) 2019

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 Cells expressing TAP-tagged Cdc16 were arrested at G1 with 1 μg/ml α-factor for 3 h and released into YPD. APC/C was immunoprecipitated at the indicated time points and western blotted for Cdh1 (upper) and Cdc20 (lower). Uncroppped western blots are provided in the Source Data file. b This schematic illustrates our APC/C-activator-binding assay. cdh1Δ APC/C (TAP-tagged on the Cdc16 subunit) was immunopurified on magnetic beads and incubated with ³⁵S-Cdc20 or ³⁵S-Cdh1 produced by translation in vitro. After removing unbound activators by washing, buffer or yeast lysate (2.5 mg/ml protein concentration) was added in the presence of 5 mM ATP. At various times, APC/C was pulled down from the reaction mix and the amount of bound activator determined by SDS–PAGE and PhosphorImager analysis. Due to the extreme dilution of APC/C-activator complexes on the beads, rebinding of dissociated activator is expected to be negligible under these conditions. c As described in panel b, we measured dissociation of radiolabeled Cdc20 (top) or Cdh1 (bottom) from immobilized APC/C over the indicated time course (at 25 °C). Buffer control contains lysis buffer and 5 mM ATP. Fraction remaining was measured by calculating the ratio of activator signal at indicated time points to zero time point signal. Results were plotted and fitted to an exponential one phase decay equation in GraphPad Prism. Similar results were observed in several independent experiments. Source data are provided in the Source Data file. d Cdh1 dissociation reactions were performed as in panel c in the presence of serially diluted yeast lysate, supplemented with 5 mM ATP. Remaining activator after 45 min is plotted as a function of protein concentration. Data indicate means (±SEM) from three independent experiments. Source data are provided in the Source Data file. e Yeast lysates were subjected to gel filtration in lysis buffer to remove molecules smaller than ~5 kDa. Desalted lysates were supplemented with buffer (black), 5 mM ATP (red), or 5 mM AMP-PNP (blue) prior to the Cdh1 dissociation reaction. These results are representative of multiple experiments. Source data are provided in the Source Data file.