Extended Data Fig. 3: Importin α1 mediates nuclear translocation of HDAC6 via SE14 domain.

a, Effects of valine deprivation on the subcellular localization of histone deacetylases. Cell fractionation analysis was performed with HCT116 cells transfected with Flag-HDAC4, Flag-HDAC6 or Flag-SIRT7. b, Effects of valine deprivation on the acetylation of HDAC6. HEK293T cells transfected with Flag-HDAC6 and HA-P300 were subjected to immunoprecipitation with Flag antibody after valine withdraw for 6 h, followed by immunoblotting analysis. c, Effects of importin inhibitor on the subcellular localization of HDAC6. Cell fractionation analysis was performed with HCT116 cells pretreated with Importazole (10 μM) for 2 h and then subjected to valine withdraw or not for 6 h. d, Immunofluorescence analysis after HCT116 cells expressing HDAC6 or its mutants were pretreated with the nuclear exportin inhibitor Leptomycin B (10 μM) or nuclear importin inhibitor Importazole (10 μM) for 2 h and then subjected to valine deprivation or not for 6 h. Scale bar, 10 μm. e, The interaction between HDAC6 and importins. Immunoblot results of cell lysates and anti-Flag or HA immunoprecipitates from HEK293T cells transfected with the indicated Flag–importin α or HA-importin β for 24 h and then subjected to valine deprivation for 6 hrs. f, Effects of valine deprivation on the interaction between hHDAC6 and importin α proteins. Immunoprecipitation was performed using HEK293T cells transfected with Flag-importin α for 24 h and subjected to valine deprivation for 6 h. g, Effects of valine re-introduction on the interaction between HDAC6 and importin α1. Immunoprecipitation was performed using HEK293T cells transfected with Flag-importin α1 for 24 h with or without re-supplement of valine for 6 h after valine deprivation for 6 h. Valine (10 mM) was add to the cell exacts before co-immunoprecipitation. h, Immunoprecipitation was performed using HEK293T cells transfected with Myc-importin α1 and Flag-HDAC6 truncation mutants (ΔNLS and ΔSE14) for 24 h and subjected to valine deprivation for 6 h. i, SE14 repeat domain only exists in primates. Multiple alignment of HDAC6 protein sequences across species was analysed by Clustal omega. Seven SE14 repeats are boxed. SE14-3 repeat is also indicated. j, Human, but not murine, HDAC6 binds to biotinylated valine. Pull-down assay was performed using HEK293T cells and MEF cells, respectively. k, Effects of valine deprivation on the subcellular localization of hHDAC6 and mHdac6. Cell fractionation analysis was performed using HCT116 cells transfected with Flag-hHDAC6 and Flag-mHdac6. l, Binding of biotinylated valine to human HDAC6, murine Hdac6 (mHdac6) or the chimera of mHdac6 and the SE14 repeat domain (cHdac6). Pull-down assay was performed using HCT116 cells transfected with Flag-hHDAC6, HA-mHdac6 and its chimaera HA-cHdac6. m, Schematic diagram of SE14 repeat domain knocking-in into the mHdac6 gene. n, The SE14 repeat domain inserted into the 25th exon of mHdac6 was verified by DNA sequencing. o, p, Validation of HDAC6 knockout efficiency via immunofluorescence (o) and immunoblotting (p). Scale bars, 10 μm. q, r, The function of SE14 repeat domain in sensing valine deprivation. The location of mHdac6 in wildtype (WT) and SE14 repeat domain knock-in (KI) MEF cells exposed to valine deprivation for 6 hrs or not were detected by cell fractionation assay (q) and immunofluorescence (r). Scale bar, 10 μm. s, The interaction between valine and the SE14 repeat domain of human and Rhinopithecus bieti. These protein of SE14 repeat domain were prepared from E. coli. t, Analysis of the subcellular location of Hdac6 in Rhinopithecus bieti-fibroblast cells via Immunofluorescence. Scale bar, 10 μm. For gel source data, see Supplementary Fig. 1.