Journal: Plant Physiology

Article Title: Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

doi: 10.1093/plphys/kiad406

Figure Lengend Snippet: Proteolytic turnover of At SQE by At DOA10A and At NAA20 in heterologous yeast degradation assays. A) Anti-HA immunoblot showing steady-state levels of AtSQE1-HA in WT vs Scdoa10Δ yeast cells. Anti-ACTIN bands are shown on the same blot. CBB, Coomassie Brilliant Blue loading control. B) Steady-state protein (immunoblot) and mRNA (RT-PCR) levels of AtSQE1-HA expressed in WT vs Scdoa10Δ ± co-expression with At DOA10A or At DOA10B. C) Cycloheximide (CHX) chase of At SQE1-HA in WT, Scdoa10Δ , and Scnaa20Δ yeast cells (immunoblot and quantified relative density). D) Cycloheximide chase showing that co-expression of At DOA10A destabilizes At SQE1-HA in Scdoa10Δ yeast cells (immunoblot and quantified relative density). E) Cycloheximide chase showing that co-expression of At NAA20 destabilizes At SQE1-HA in Scnaa20Δ yeast cells (immunoblot and quantified relative density).

Article Snippet: Yeast were transformed with AtDOA10A , AtDOA10B , and AtNAA20 (At1g03150) in the pAG416GPD-ccdB-EGFP vector (Addgene plasmid #14196, Susan Lindquist) and AtSQE1 in the pAG413GPD-ccdB-HA vector (Addgene plasmid #14238, Susan Lindquist) or the corresponding empty vectors.

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Plant Physiology

Article Title: Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

doi: 10.1093/plphys/kiad406

Figure Lengend Snippet: Impact of N-terminal mutagenesis on AtSQE1 stability suggests indirect effects of Nt-acetyltransferases on protein turnover in yeast. A) Summary of N-terminal (Nt) mutants and predicted respective NAT activities. B) Steady-state protein levels of At SQE1-HA Nt-variants in WT and Scdoa10Δ yeast cells. C) Cycloheximide (CHX) chase of WT ME- and mutant MP- At SQE1-HA in WT yeast cells (immunoblot and quantified relative density). D) Cycloheximide (CHX) chase of WT ME- At SQE1-HA in WT and Scnaa10Δ yeast cells (immunoblot and quantified relative density). E) Cycloheximide (CHX) chase of mutant MA- At SQE1-HA in WT and Scnaa20Δ yeast cells (immunoblot and quantified relative density). F) Cycloheximide (CHX) chase of mutant MA- At SQE1-HA in WT and Scnaa10Δ yeast cells (immunoblot and quantified relative density).

Article Snippet: Yeast were transformed with AtDOA10A , AtDOA10B , and AtNAA20 (At1g03150) in the pAG416GPD-ccdB-EGFP vector (Addgene plasmid #14196, Susan Lindquist) and AtSQE1 in the pAG413GPD-ccdB-HA vector (Addgene plasmid #14238, Susan Lindquist) or the corresponding empty vectors.

Techniques: Mutagenesis, Western Blot

Journal: Plant Physiology

Article Title: Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases

doi: 10.1093/plphys/kiad406

Figure Lengend Snippet: Summary of the mevalonate (MVA) and sterol synthesis pathways in Arabidopsis : (1) DOA10 negatively regulates SQE1 stability (this study) and positively regulates HMGR activity in plants, yeast, and humans. Yeast and animal SQEs were previously shown to be targets of DOA10 ( ; ), indicating conservation of this regulatory module across three eukaryotic kingdoms. (2) NATA and B were shown to indirectly contribute to AtSQE1 turnover in yeast (this study), but not in Arabidopsis . (3) LDAO, an inhibitor of several downstream enzymatic steps, also negatively regulates AtSQE1 levels via DOA10-independent mechanism(s). (4) Terbinafine, a chemical inhibitor of SQE enzymatic activity , indirectly promotes accumulation of AtSQE1 (this study), likely through positive feedback. Dashed lines denote indirect effects.

Article Snippet: Yeast were transformed with AtDOA10A , AtDOA10B , and AtNAA20 (At1g03150) in the pAG416GPD-ccdB-EGFP vector (Addgene plasmid #14196, Susan Lindquist) and AtSQE1 in the pAG413GPD-ccdB-HA vector (Addgene plasmid #14238, Susan Lindquist) or the corresponding empty vectors.

Techniques: Activity Assay

Journal: Plant physiology

Article Title: Nt-acetylation-independent turnover of SQUALENE EPOXIDASE 1 by Arabidopsis DOA10-like E3 ligases.

doi: 10.1093/plphys/kiad406

Figure Lengend Snippet: Figure 5. Proteolytic turnover of AtSQE by AtDOA10A and AtNAA20 in heterologous yeast degradation assays. A) Anti-HA immunoblot showing steady-state levels of AtSQE1-HA in WT vs Scdoa10Δ yeast cells. Anti-ACTIN bands are shown on the same blot. CBB, Coomassie Brilliant Blue load ing control. B) Steady-state protein (immunoblot) and mRNA (RT-PCR) levels of AtSQE1-HA expressed in WT vs Scdoa10Δ ± co-expression with AtDOA10A or AtDOA10B. C) Cycloheximide (CHX) chase of AtSQE1-HA in WT, Scdoa10Δ, and Scnaa20Δ yeast cells (immunoblot and quantified relative density). D) Cycloheximide chase showing that co-expression of AtDOA10A destabilizes AtSQE1-HA in Scdoa10Δ yeast cells (immunoblot and quantified relative density). E) Cycloheximide chase showing that co-expression of AtNAA20 destabilizes AtSQE1-HA in Scnaa20Δ yeast cells (immunoblot and quantified relative density).

Article Snippet: Yeast were transformed with AtDOA10A, AtDOA10B, and AtNAA20 (At1g03150) in the pAG416GPD-ccdB-EGFP vector (Addgene plasmid #14196, Susan Lindquist) and AtSQE1 in the pAG413GPD-ccdB-HA vector (Addgene plasmid #14238, Susan Lindquist) or the corresponding empty vectors.

Techniques: Western Blot, Control, Reverse Transcription Polymerase Chain Reaction, Expressing