Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome with syringolin A

Abstract

As proteins are the main effectors inside cells, their levels need to be tightly regulated. This is partly achieved by specific protein degradation via the Ubiquitin-26S proteasome system (UPS). In plants, an exceptionally high number of proteins are involved in Ubiquitin-26S proteasome system-mediated protein degradation and it is known to regulate most, if not all, important cellular processes. Here, we investigated the response to the inhibition of the proteasome at the protein level treating leaves with the specific inhibitor Syringolin A (SylA) in a daytime specific manner and found 109 accumulated and 140 decreased proteins. The patterns of protein level changes indicate that the accumulating proteins cause proteotoxic stress that triggers various responses. Comparing protein level changes in SylA treated with those in a transgenic line over-expressing a mutated ubiquitin unable to form polyubiquitylated proteins produced little overlap pointing to different response pathways. To distinguish between direct and indirect targets of the UPS we also enriched and identified ubiquitylated proteins after inhibition of the proteasome, revealing a total of 1791 ubiquitylated proteins in leaves and roots and 1209 that were uniquely identified in our study. The comparison of the ubiquitylated proteins with those changing in abundance after SylA-mediated inhibition of the proteasome confirmed the complexity of the response and revealed that some proteins are regulated both at transcriptional and post-transcriptional level. For the ubiquitylated proteins that accumulate in the cytoplasm but are targeted to the plastid or the mitochondrion, we often found peptides in their target sequences, demonstrating that the UPS is involved in controlling organellar protein levels. Attempts to identify the sites of ubiquitylation revealed that the specific properties of this post-translational modification can lead to incorrect peptide spectrum assignments in complex peptide mixtures in which only a small fraction of peptides is expected to carry the ubiquitin footprint. This was confirmed with measurements of synthetically produced peptides and calculating the similarities between the different spectra.

Fig. 1.

Accumulation of ubiquitylated proteins after treatment with SylA. Western blot of protein samples isolated from SylA treated and control leaves using the α-ubiquitin-K48 antibody (top) and the α-tubulin antibody (bottom, loading control). The membrane sections probed with the two antibodies were obtained from the same gel. The samples loaded were from three different biological replicates of leaves either treated with 10 μm SylA in Tween20 (SylA1, SylA2, and SylA3) or with Tween20 only (Twe1, Twe2, and Twe3) at the end of the night and harvested 8 h later at the end of the day (see Fig. 3). The molecular weight is indicated on the left.

Fig. 2.

Workflow for the identification of proteins changing in abundance after proteasome inhibition by treatment with SylA. Before the onset of light, the leaves were treated with SylA or Tween20 only. At the same time, a T0 control was harvested (left). After 8 h of light exposure, the ED leaf sample was harvested. At the same time point, leaves were treated with SylA or Tween20 only and an untreated ED T0 control was harvested. After further growth for 8 h and 16 h in the dark the MN and EN samples were harvested, respectively.

Fig. 3.

Western blot (A) using the α-HSP90 antibody (top) and α-tubulin antibody (bottom, loading control) and (B) for α-POR antibody (bottom) and α-DPE2 antibody (top, loading control). The samples loaded were from three different biological replicates of leaves from plants either treated with 10 μm SylA in Tween20 or with Tween20 only (Twe1, Twe2 and Twe3). At the left, the molecular weight is indicated.

Fig. 4.

Different possible scenarios for protein level changes observed after SylA treatment. A, The protein levels in the SylA treated sample are significantly higher than in the mock treated sample and in the T0 sample at the time of treatment. B, The protein levels in the mock treated and the untreated sample T0 at the time of harvest are significantly lower than in the SylA treated sample and the T0 sample at the time of treatment. C, The protein levels in the mock treated and the T0 sample at the time of harvest are significantly higher as compared with the SylA treated sample and the T0 sample at the time of treatment. D, The protein levels in the SylA treated sample are significantly lower than in the mock treated sample and the T0 sample at the time of treatment without a significant change between the mock treated and untreated sample in comparison to the T0 sample at the time of treatment.

Fig. 5.

Overlap of reported ubiquitylated proteins in seedlings (Manzano et al., (23); Igawa et al., (24); Saracco et al., (25), and Kim et al., (26)) and cell culture (Maor et al., (22)) with the proteins from leaves and roots in this study (A) and with the proteins identified in the two serial native and urea extracts of leaves (B) and roots (C).

Fig. 6.

Frequencies of the dot product correlations between the different spectra, which provide a quantitative measure for the similarity between two spectra. The higher the dot product, the more similar are the spectra in a pairwise comparison. The frequencies were calculated for intervals of 0.05.