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Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.

Svozil J, Gruissem W, Baerenfaller K - Front Plant Sci (2015)

Bottom Line: SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins.Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated.Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Department of Biology, Swiss Federal Institute of Technology Zurich Zurich, Switzerland.

ABSTRACT
Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

No MeSH data available.


Schematic overview of the aliphatic glucosinolate biosynthesis consisting of methionine chain elongation (A), glucone formation (B) and side chain modification (C), displaying the proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue extracts (green), that were exclusively identified as ubiquitylated in the vascular but not in the epidermal or mesophyll tissue extracts (blue), or both (red). In brown the proteins that were not exclusively identified in vasculature and in gray those that were not identified.
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Figure 5: Schematic overview of the aliphatic glucosinolate biosynthesis consisting of methionine chain elongation (A), glucone formation (B) and side chain modification (C), displaying the proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue extracts (green), that were exclusively identified as ubiquitylated in the vascular but not in the epidermal or mesophyll tissue extracts (blue), or both (red). In brown the proteins that were not exclusively identified in vasculature and in gray those that were not identified.

Mentions: In vascular tissue protein extracts we identified an exceptionally high number of 353 ubiquitylated proteins (Table 1). Apart from various response pathways, the GO category glucosinolate biosynthetic process was over-represented in this set of proteins. This category was also over-represented in the list of proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue protein extracts (Table 4). Glucosinolates are derived from different amino acids, including methionine and phenylalanine. Depending on the amino acid precursor, they are assigned to different groups. Here, we focus on the biosynthesis of aliphatic glucosinolates, since the identified ubiquitylated proteins mainly catalyze reactions in this pathway. The biosynthesis of aliphatic glucosinolates involves three major steps (Grubb and Abel, 2006; Sawada et al., 2009; S√łnderby et al., 2010). The first step is methionine chain elongation, which includes methionine deamination, repeated condensation, isomerization, oxidative decarboxylation and transamination. The proteins involved in this step were exclusively identified in vascular tissue protein extract and in affinity enriched ubiquitylated proteins from vascular tissue (Table 4, Figure 5). In the second step the glucone core structure is formed, which involves the incorporation of sulfur. All of the identified enzymes of the core biosynthetic process except for GGP1 were also identified exclusively in the vascular tissue protein extract or after affinity purification (Table 4, Figure 5). The third and last step in glucosinolate biosynthesis is secondary side chain modification. The identified enzymes in this process were found to be ubiquitylated exclusively in the vasculature tissue (Figure 5; Table 4). In summary, we identified most of the enzymes that catalyze the core biosynthetic steps of glucosinolate biosynthesis only in vascular tissue extracts and in affinity-enriched ubiquitylated proteins from the vascular tissue.


Proteasome targeting of proteins in Arabidopsis leaf mesophyll, epidermal and vascular tissues.

Svozil J, Gruissem W, Baerenfaller K - Front Plant Sci (2015)

Schematic overview of the aliphatic glucosinolate biosynthesis consisting of methionine chain elongation (A), glucone formation (B) and side chain modification (C), displaying the proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue extracts (green), that were exclusively identified as ubiquitylated in the vascular but not in the epidermal or mesophyll tissue extracts (blue), or both (red). In brown the proteins that were not exclusively identified in vasculature and in gray those that were not identified.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4446536&req=5

Figure 5: Schematic overview of the aliphatic glucosinolate biosynthesis consisting of methionine chain elongation (A), glucone formation (B) and side chain modification (C), displaying the proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue extracts (green), that were exclusively identified as ubiquitylated in the vascular but not in the epidermal or mesophyll tissue extracts (blue), or both (red). In brown the proteins that were not exclusively identified in vasculature and in gray those that were not identified.
Mentions: In vascular tissue protein extracts we identified an exceptionally high number of 353 ubiquitylated proteins (Table 1). Apart from various response pathways, the GO category glucosinolate biosynthetic process was over-represented in this set of proteins. This category was also over-represented in the list of proteins that were exclusively identified in the vascular but not in the epidermal or mesophyll tissue protein extracts (Table 4). Glucosinolates are derived from different amino acids, including methionine and phenylalanine. Depending on the amino acid precursor, they are assigned to different groups. Here, we focus on the biosynthesis of aliphatic glucosinolates, since the identified ubiquitylated proteins mainly catalyze reactions in this pathway. The biosynthesis of aliphatic glucosinolates involves three major steps (Grubb and Abel, 2006; Sawada et al., 2009; S√łnderby et al., 2010). The first step is methionine chain elongation, which includes methionine deamination, repeated condensation, isomerization, oxidative decarboxylation and transamination. The proteins involved in this step were exclusively identified in vascular tissue protein extract and in affinity enriched ubiquitylated proteins from vascular tissue (Table 4, Figure 5). In the second step the glucone core structure is formed, which involves the incorporation of sulfur. All of the identified enzymes of the core biosynthetic process except for GGP1 were also identified exclusively in the vascular tissue protein extract or after affinity purification (Table 4, Figure 5). The third and last step in glucosinolate biosynthesis is secondary side chain modification. The identified enzymes in this process were found to be ubiquitylated exclusively in the vasculature tissue (Figure 5; Table 4). In summary, we identified most of the enzymes that catalyze the core biosynthetic steps of glucosinolate biosynthesis only in vascular tissue extracts and in affinity-enriched ubiquitylated proteins from the vascular tissue.

Bottom Line: SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins.Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated.Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

View Article: PubMed Central - PubMed

Affiliation: Plant Biotechnology, Department of Biology, Swiss Federal Institute of Technology Zurich Zurich, Switzerland.

ABSTRACT
Protein and transcript levels are partly decoupled as a function of translation efficiency and protein degradation. Selective protein degradation via the Ubiquitin-26S proteasome system (UPS) ensures protein homeostasis and facilitates adjustment of protein abundance during changing environmental conditions. Since individual leaf tissues have specialized functions, their protein composition is different and hence also protein level regulation is expected to differ. To understand UPS function in a tissue-specific context we developed a method termed Meselect to effectively and rapidly separate Arabidopsis thaliana leaf epidermal, vascular and mesophyll tissues. Epidermal and vascular tissue cells are separated mechanically, while mesophyll cells are obtained after rapid protoplasting. The high yield of proteins was sufficient for tissue-specific proteome analyses after inhibition of the proteasome with the specific inhibitor Syringolin A (SylA) and affinity enrichment of ubiquitylated proteins. SylA treatment of leaves resulted in the accumulation of 225 proteins and identification of 519 ubiquitylated proteins. Proteins that were exclusively identified in the three different tissue types are consistent with specific cellular functions. Mesophyll cell proteins were enriched for plastid membrane translocation complexes as targets of the UPS. Epidermis enzymes of the TCA cycle and cell wall biosynthesis specifically accumulated after proteasome inhibition, and in the vascular tissue several enzymes involved in glucosinolate biosynthesis were found to be ubiquitylated. Our results demonstrate that protein level changes and UPS protein targets are characteristic of the individual leaf tissues and that the proteasome is relevant for tissue-specific functions.

No MeSH data available.