Fig 2: Depletion of NatA activity causes enhanced degradation of proteins via the ubiquitin-proteasome system.a Time-resolved analysis of protein degradation rate in leaves of wild type (black) and four individual lines depleted for the ribosome anchoring (NAA15, light green) or the catalytically active subunit (NAA10, dark green) of the NatA complex. n = 4 biologically independent samples, except for WT 360 min, amiNaa10 # 18 360 min, amiNaa10 #23 120 min, 240 min, amiNaa15 #8 120 min, 360 min, and amiNaa15 #10 360 min n = 3. b Proteasome activity in wild type and NatA activity depleted plants. Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak, one-way ANOVA (p < 0.05, n = 4 biologically independent samples, except for amiNaa10 #18 n = 3). c Relative level of poly-ubiquitinated proteins as determined with the ubiquitin-specific antiserum (a-UBQ11; Agrisera) in leaves of wild type and NatA depleted plants in the presence (red) or absence (black) of the proteasome inhibitor MG132. Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak, one-way ANOVA (p < 0.05, n = 3 biologically independent samples). d Abundance and activation status of Cullin-RING E3 ligase (CRL) complexes as determined by neddylation of Cullin isoform 1 in wild type and transgenic lines depleted of NAA15 (muse6) and NAA10 (amiNAA10). Different letters indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak, one-way ANOVA (p < 0.05, n = 3 biologically independent samples) e Immunological detection of poly-ubiquitinated proteins using an ubiquitin-specific antibody (a-UBQ11; Agrisera) in the wild type and amiNAA10 line 23 (amiNAA10) after selective enrichment with the Ubi-Qapture QTM matrix. f Protein amount of affinity enriched bona fide poly-ubiquitinated proteins. Asterix indicate individual groups identified by pairwise multiple comparisons with a Holm-Sidak, one-way ANOVA (p < 0.05, n = 3 biologically independent samples) g Quantitative proteomics of bona fide poly-ubiquitinated proteins revealed that 24% of quantified proteins (232 proteins) were significantly (student t-test, p < 0.05) more ubiquitinated in amiNAA10 (>1.5-fold more than wild type) or either only found in all amiNAA10 replicates (n = 3 biologically independent samples). The pie chart depicts the classification of Nat substrates in the fraction of significantly more ubiquitinated proteins in amiNAA10 plants. Data are shown as mean ± standard error. Immunological detections of proteins quantified in c, d were independently repeated and blots are shown in the source data file.

Fig 3: Effect of T264 phosphorylation on SR45 ubiquitination and degradation.(A) Protein gel blot analysis of the SR45–GFP fusion protein immunoprecipitated from extracts of 7-day-old seedlings of the OX1 overexpression (pUBQ10:SR45–GFP/sr45-1), PMut1 phosphomutant (pUBQ10:SR45–GFP_T264A/sr45-1), and PMim1 phosphomimetic (pUBQ10:SR45–GFP_T264D/sr45-1) transgenic lines grown under control conditions using a-GFP (immunoprecipitation; IP) or a-UBQ11 (coIP) antibodies. Ponceau staining is shown as a loading control for the input fraction. Signals were quantified and the UBQ/SR45–GFP ratio determined, with control conditions set to 1. Results represent means ± SE (n = 3), and different letters indicate statistically significant differences between genotypes (P < 0.05; Student’s t-test).(B) Protein gel blot analysis, using a-GFP antibodies, of the SR45–GFP fusion protein in 7-day-old seedlings of the OX1 overexpression, PMut1 phosphomutant, and PMim1 phosphomimetic transgenic lines supplemented or not with MG132 and left at room temperature for 0, 15, 30, or 60 min. Control samples (-MG132) were treated with the equivalent volume of the solvent of the MG132 solution (DMSO), and a total of 20 ng of protein was loaded per sample. Bands were quantified and relative protein levels determined using the Ponceau loading control as a reference, with time 0 set to 1. Results are representative of at least three independent experiments.

Fig 4: Effect of ABA on SR45 protein stability and ubiquitination.(A) Protein gel blot analysis, using a-GFP antibodies, of the SR45–GFP fusion protein in 7-day-old seedlings of the C2 complementation line pretreated with MG132 and subjected to a 180-min treatment with 2 µM ABA. Control samples (-MG132 or -ABA) were treated with the equivalent volume of the solvent of the MG132 or ABA solution (DMSO or ethanol, respectively). A total of 40 ng of protein was loaded per sample. Bands were quantified and relative protein levels determined using the Ponceau loading control as a reference, with control conditions set to 1. Results represent means ± SE (n = 4), and different letters indicate statistically significant differences between treatments (P < 0.05; Student’s t-test).(B) Protein gel blot analysis of the SR45–GFP fusion protein immunoprecipitated from extracts of 7-day-old seedlings of the C2 complementation line treated for 180 min with 2 µM ABA using a-GFP (immunoprecipitation; IP) or a-UBQ11 (coIP) antibodies. Control samples were treated with the equivalent volume of the solvent of the ABA solution (ethanol). Ponceau staining is shown as a loading control for the input fraction. Signals were quantified and the UBQ/SR45–GFP ratio determined, with control conditions set to 1. Results represent means ± SE (n = 3), and different letters indicate statistically significant differences between treatments (P < 0.05; Student’s t-test).