Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase

Bhuiyan, Nazmul H.; Rowland, Elden; Friso, Giulia; Ponnala, Lalit; Michel, Elena J.S.; Wijk, Klaas J. van

doi:10.1104/pp.20.00752

Cited by 8 publications

(4 citation statements)

References 69 publications

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“…We prepared fusion constructs encoding the C-terminal fragment of the yellow fluorescent protein (cYFP) fused to full-length mature DUF760-1 (DUF760-1-cYFP), DUF760-2 (DUF760-2-cYFP) or mature CGEP-S781R (CGEP-S781R-cYFP) under the control of the 35S promoter. CGEP-S781R is a catalytically inactive chloroplast glutamyl aminopeptidase that operates independently of the CLP chaperone-protease complex (Bhuiyan et al, 2020) and thus serves as a negative control for CLPC1 interactions. Additionally, we generated fusion constructs encoding the N-terminal fragment of YFP (nYFP) fused to the full-length CGEP-S781R (CGEP-S781R-nYFP), CLPC1 (CLPC1-nYFP) or CLPC1-TRAP (CLPC1-TRAP-nYFP).…”

Section: Resultsmentioning

confidence: 99%

Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

Yuan,

van Wijk

2024

Preprint

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The chloroplast AAA+ chaperone CLPC1 aids to select, unfold and deliver hundreds of proteins to the stromal CLP protease core complex for degradation. Through in vivo CLPC1 trapping we previously identified dozens of trapped proteins that are substrate adaptors (e.g. CLPS1 and CLPF), other chaperones (CLPC2 and CLPD) or (potential) substrates (e.g. RH3) for the CLP chaperone-protease system. Here we show that two of these highly trapped proteins, DUF760-1 and DUF760-2, are substrates for the chloroplast CLP protease system. In planta BiFC and yeast-2-hybrid analyses show that both DUF760 proteins can directly interact with the N-domain of CLPC1. Immunoblotting and confocal microscopy analysis demonstrates that both DUF760 proteins are highly enriched in clpc1-1 and clpr2-1 loss-of function mutants. In vivo cycloheximide chase assays in different genetic backgrounds show that DUF760-1 and 2 are both degraded by the chloroplast CLP protease. The half-life of DUF760-1 is 4-6 hours, whereas DUF760-2 is so unstable that it is very hard to detect unless degradation is inhibited in CLP loss-of-function alleles. Null mutants for DUF760-1 and DUF760-2 show weak but differential pigment phenotypes and differential sensitivity of protein translation inhibitors. The functions of these DUF760 proteins are unknown; the lack of shared mRNA co-expressors and the large difference in half-life and protein abundance, suggest that they play different roles within the chloroplast. Taken together, our results demonstrate that DUF760-1 and 2 are newly discovered substrates of CLP chaperone-protease system.

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Section: Resultsmentioning

confidence: 99%

Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

Yuan,

van Wijk

2024

Preprint

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“…These plastid proteins represented ∼72% of the protein biomass based on both adjusted Spectral Counts (adjSPC) and normalized adjSPC (NadjSPC). Previously, we also carried out a similar in vivo protein interaction analysis for transgenic plants expressing two different STREPII-tagged versions of the unrelated chloroplast glutamyl peptidase CGEP ( 13 ). As described ( 13 ), this did not identify any strong candidate interactors to CGEP, and this dataset therefore serves as an excellent negative control for nonspecific binding to the streptactin affinity columns and for abundant proteins.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, we also carried out a similar in vivo protein interaction analysis for transgenic plants expressing two different STREPII-tagged versions of the unrelated chloroplast glutamyl peptidase CGEP ( 13 ). As described ( 13 ), this did not identify any strong candidate interactors to CGEP, and this dataset therefore serves as an excellent negative control for nonspecific binding to the streptactin affinity columns and for abundant proteins. Proteins also identified in the CGEP-STREP affinity experiments are listed in Table S1 .…”

Section: Resultsmentioning

confidence: 99%

Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

Liao

Friso²,

Forsythe³

et al. 2022

Journal of Biological Chemistry

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This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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“…Furthermore, a good confirmation that we successfully enriched plastids from vascular tissue came from the higher abundance of CGEP, a glutamyl peptidase part of the chloroplast proteostasis network. This peptidase, which cleaves after glutamate residues ( Bhuiyan et al , 2020 ), was reported to be in 4-fold higher abundance in chloroplasts of bundle-sheath cells as compared with mesophyll cells in maize ( Friso et al , 2010 ). That said, the vast majority of proteins that were found in a significantly higher abundance were in chloroplasts from vascular cells.…”

Section: Discussionmentioning

confidence: 99%

Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells

Boussardon

Carrie

Keech

2023

Journal of Experimental Botany

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Plastids are complex organelles that vary in size and function depending on the cell type. Accordingly, they can be referred to as amyloplasts, chloroplasts, chromoplasts, etioplasts, proplasts to only cite a few denominations. Over the past decades, methods based on density gradients and differential centrifugations have been extensively used for the purification of plastids. However, these methods need large amounts of starting material, and hardly provide a tissue-specific resolution. Here, we applied our IPTACT (Isolation of Plastids TAgged in specific Cell Types) method, which involves the biotinylation of plastids in vivo using one-shot transgenic lines expressing the TOC64 gene coupled with a biotin ligase receptor particle and the BirA biotin ligase, to isolate plastids from mesophyll and companion cells of Arabidopsis thaliana using tissue specific pCAB3 and pSUC2 promoters, respectively. Subsequently, a proteome profiling was performed, and allowed the identification of 1672 proteins, among which 1342 were predicted plastidial, and 705 were fully confirmed according to SUBA5. Interestingly, although 92% of plastidial proteins were equally distributed between the two tissues, we observed an accumulation of proteins associated with jasmonic acid biosynthesis, plastoglobuli (e.g. NDC1, VTE1, PGL34, ABC1K1) and cyclic electron flow in plastids originating from vascular tissues. Besides demonstrating the technical feasibility of isolating plastids in a tissue-specific manner, our work provides strong evidence that plastids from vascular tissue have a higher redox turnover to ensure optimal functioning, notably under high solute strength as encountered in vascular cells.

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Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase

Abstract: Auto-catalytic processing and substrate specificity of Arabidopsis chloroplast glutamyl peptidase

Cited by 8 publications

References 69 publications

Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

Stromal DUF760-1 and DUF760-2 proteins are degraded by the chloroplast Clp protease system but have very different half-lives

Proteomics, phylogenetics, and coexpression analyses indicate novel interactions in the plastid CLP chaperone-protease system

Comparison of plastid proteomes points towards a higher plastidial redox turnover in vascular tissues than in mesophyll cells

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