Degradation of the COPI proteins Ret1 and Sec21 by the AID system.

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Degradation of the COPI proteins Ret1 and Sec21 by the AID system. Degradation of the COPI proteins Ret1 and Sec21 by the AID system. (A) Expression of IAA17-tagged Ret1 and Sec21 from low-copy plasmids rescued the temperature-sensitive phenotypes of ret1-1 and sec21-1 or sec21-2 cells, respectively. (B) Growth of wild-type (WT), AtTIR1, Ret1-IAA17, Ret1-aid, Sec21-IAA17 and Sec21-aid cells on a YPD plate with or without 0.5 mM NAA at 30°C. (C) Growth of NAA-treated cells after NAA washout. Wild-type, Ret1-aid and Sec21-aid cells were cultured in YPD medium with or without 1 mM NAA for 2 h at 30°C. After washing out medium with water three times, cells were grown on a YPD plate at 30°C. (D) Ret1-aid and Sec21-aid cells were cultured in YPD medium with or without 1 mM NAA at 30°C. Cells were collected at 0, 1 and 2 h after NAA addition. Ret1-IAA17, Sec21-IAA17 and CPY proteins were detected by immunoblotting. The positions of the ER form (p1), Golgi form (p2) and mature (m) CPY are indicated on the right. The PGK lane shows the loading control. All experiments were performed three times and a representative example shown. Midori Ishii et al. J Cell Sci 2016;129:3251-3261 © 2016. Published by The Company of Biologists Ltd