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Characterisation of the free and membrane-associated fractions of the thylakoid lumen proteome in Arabidopsis thaliana

ABSTRACT: In plants chloroplasts, the lumen of the thylakoid membrane houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used gel- and mass spectrometry-based proteomics methods to analyse the contents of each proteome. These results identified lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support of the oxygen-evolving complex (OEC). Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several potential multi-protein oligomers in the thylakoid lumen.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Leaf

SUBMITTER: Andrea Trotta

LAB HEAD: Eva-Mari Aro

PROVIDER: PXD027229 | Pride | 2021-07-30

REPOSITORIES: Pride

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			Action	DRS
	2D_Lumen_1.msf	Msf
	2D_Lumen_1.mzML	Mzml
	2D_Lumen_1.mzid.gz	Mzid
	2D_Lumen_1.raw	Raw
	2D_Lumen_10.msf	Msf

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Quantification of the ratio between the two PsbO protein isoforms in the thylakoids lumen of Arabidopsis thaliana

Project description:In plants chloroplasts, the lumen of the thylakoid membrane houses proteins that are vital for photosynthetic electron transport, including water-splitting at photosystem (PS) II and shuttling of electrons from cytochrome b6f to PSI. Other lumen proteins maintain photosynthetic activity through biogenesis and turnover of PSII complexes. Although all lumen proteins are soluble, these known details have highlighted interactions of some lumen proteins with thylakoid membranes or thylakoid-intrinsic proteins. Meanwhile, the functional details of most lumen proteins, as well as their distribution between the soluble and membrane-associated lumen fractions, remain unknown. The current study isolated the soluble free lumen (FL) and membrane-associated lumen (MAL) fractions from Arabidopsis thaliana, and used mass spectrometry-based proteomics methods to analyse the contents of each proteome. These results identified lumenal proteins, and clearly distinguished the difference between the FL and MAL proteomes. The most abundant proteins in the FL fraction were involved in PSII assembly and repair, while the MAL proteome was enriched in proteins that support of the oxygen-evolving complex (OEC). Selected Reaction Monitoring (SRM) was employed to assess the ratio bewteen PsbO1 and PsbO2 in FL and MAL proteomes. Novel proteins, including a new PsbP domain-containing isoform, as well as several novel post-translational modifications and N-termini, are reported, and bi-dimensional separation of the lumen proteome identified several potential multi-protein oligomers in the thylakoid lumen.

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Formation of ER-lumenal intermediates during export of Plasmodium proteins containing transmembrane-like hydrophobic sequences

Project description:REX3RQLSE:Pf332:C-S11:DSLE is exported and is diffusely localised in the red blood cell cytoplasm (Fig 3A). Mutation of the PEXEL sequence (REX3AQLSE:Pf332:C-S11:DSLE), results in the protein being retained in the parasite (Fig 3C), and addition of a C-terminal SDEL sequence (REX3RQLSE:Pf332:C-S11:SDEL) also leads to retention of the protein within the parasite (Fig 3B). Given that REX3RQLSE:Pf332:C-S11:SDEL is retained within the parasite, this indicates that the C-terminus of the protein is in the ER lumen. To determine the location of the N-terminal end of this ER-retained protein, it was purified for tryptic digestion and analysis by mass spectrometry. The most N-terminal peptides retrieved corresponded to N-acetylated and non-acetylated SEPVVEEQDLK and SEPVVEEQDLKK. These peptides correspond to the expected N-terminal sequence following PEXEL cleavage by Plasmepsin V, indicating that the N-terminus of this protein is also in the ER-lumen. These data indicate that both N- and C-terminal ends of REX3RQLSE:Pf332:C-S11:SDEL are located within the ER lumen and consequently that in the context of this protein, the putative transmembrane segment of Pf332 has a propensity to translocate into the ER lumen rather than integrate into the ER membrane.

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Identification of allergenomic signatures in allergic and well-tolerated apple genotypes using LC-MS/MS

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2024-12-13 | PXD050814 | Pride

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Project description:The maltose regulon (mal regulon) has previously been shown to consist of the mal gene cluster (malQP, malXCD and malAR operons) in Streptococcus pneumoniae. In this study, we have further elucidated the complete mal regulon in S. pneumoniae D39 using microarray analyses and β-galactosidase assays. In addition to the mal gene cluster, the complete mal regulon of S. pneumoniae D39 consists of a pullulanase (PulA), a glucosidase (DexB), a glucokinase (RokB), a PTS component (PtsG) and an amylase (AmyA2). Our microarray studies and β-galactosidase assays further showed that the LacI-family transcriptional regulator MalR represses the expression of the mal regulon in the absence of maltose. Furthermore, the role of the pleiotropic transcriptional regulator CcpA in the regulation of the mal regulon in the presence of maltose was also explored. Our microarray analysis with a ΔccpA strain showed that CcpA only represses the expression of the malXCD operon and the pulA gene in the presence of maltose. Comparison of the Streptococcus pneumoniae D39 wild type in MM17 (0.5 % (w/v) Maltose + M17) medium compared to GM17 (0.5 % (w/v) Glucose + M17) One condition design comparision of two strains including a dye swap

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