Fig. 1: Coronavirus M protein is degraded by SQSTM1-dependent autophagy.

A Immunoblotting and immunoprecipitation of the lysates of HEK293T cells cotransfected with Flag-SQSTM1 and the indicated vectors expressing GFP tagged-PEDV M (GFP-PEDV M), GFP tagged-SARS-CoV-2 M (GFP-SARS M), GFP tagged-IBV M (GFP-IBV M), or GFP tagged-PDCoV M (GFP-PDCoV M). GFP was used as a control. B Schematic diagram of full-length or truncated SARS-CoV-2 M (GFP-M) protein structures, including various M truncated mutants that lacked the N-terminal disordered region (GFP-MΔDR1), transmembrane domain (GFP-MΔTM), conserved domain and/or β-sheets 1-5 (GFP-MΔCDβ_1-5, GFP-MΔCD, GFP-MΔβ_1-2 or GFP-MΔβ_3-5; β₁ and β₂ are outer sheets; β₃ to β₅ are inner sheets), C-terminal β-sheets 6-8 (GFP-MΔβ_6-8, GFP-MΔβ_6-7, GFP-MΔβ₈; β₆ and β₇ are outer sheets; β₈ is an inner sheet), and C-terminal disordered region (GFP-MΔDR2). C Immunoblotting and immunoprecipitation of the lysates of HEK293T cells cotransfected with Flag-SQSTM1 and the indicated vectors expressing full-length M (GFP-M) or various M truncated mutants (GFP-MΔDR1, GFP-MΔTM, GFP-MΔCDβ_1-5, GFP-MΔCD, GFP-MΔβ_1-2, GFP-MΔβ_3-5, GFP-MΔβ_6-8, GFP-MΔβ_6-7, GFP-MΔβ_8, GFP-MΔDR2). GFP was used as a control. D Schematic diagram of split GFP_1-10/11 fluorescence. When proteins fused with GFP_1-10 or GFP₁₁ interact, GFP_1-10 self-associate with GFP₁₁ to reconstitute a functional GFP. E Split GFP_1-10/11 florescence of the proteins between full length or mutant M fused with GFP_1-10 and SQSTM1 fused with GFP₁₁. F Quantification of split GFP_1-10/11 fluorescence. The split GFP_1-10/11 signal from (E) was calculated using ImageJ software. n = 6 images analyzed for each condition. G MST analysis of the indicated protein interactions. K_D measurements of SQSTM1 with different ligands (M, M-CD and MΔCD) were derived from the binding response as a function of the GFP-SQSTM1 concentration. H Immunoblotting of the lysates of HEK293T cells or SQSTM1-KO cells transefected with indicated vectors (n = 3 biological replicates). I Confocal analysis of colocalization between full length or mutant M and LC3B in presence or absence of SQSTM1. Statistical analysis was performed using unpaired two-tailed Student’s t-tests, mean ± SEMs, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NS = P not significant. Source data are provided as a Source Data file.