Thomas Kieselbach - Academia.edu (original) (raw)

Papers by Thomas Kieselbach

Photosynthesis Research, 2003

Recent research in proteomics of the higher plant chloroplast has achieved considerable progress ... more Recent research in proteomics of the higher plant chloroplast has achieved considerable progress and added to our knowledge of lumenal chloroplast proteins. This work shows that chloroplast lumen has its own specific proteome and may comprise as many as 80 proteins. Although the new map of the lumenal proteome provides a great deal of information, it also raises numerous questions because the physiological functions of most of the novel lumenal proteins are unknown. In this Minireview, we summarize the latest discoveries regarding lumenal proteins and present the currently known facts about the lumenal chloroplast proteome of higher plants.

Photosynthesis Research, 2003

Currently, relatively few proteomics studies of chloroplast have been published, but the field ha... more Currently, relatively few proteomics studies of chloroplast have been published, but the field has just started emerging and is likely to develop more rapidly in the future. While the complex membrane structure of the chloroplast makes it difficult to study its entire proteome by global approaches, proteomics has considerably increased our knowledge of the proteins of single compartments such as, for instance, the envelope and the thylakoid lumen. Proteomics has also succeeded in the subunit characterisation of select protein complexes such as the ribosomes and the cytochrome b 6 f complex. In addition, proteomics was successfully applied to find new potential target pathways for thioredoxin-mediated signal transduction. In this review, we present an overview of the latest developments in the field of chloroplast proteomics and discuss their impact on photosynthesis research. In addition, we summarise the current state of research in proteomics of the photosynthetic cyanobactrium Synechocystis sp. PCC 6803.

PloS one, 2015

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive... more Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we no...

PloS one, 2011

RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels... more RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression. In this study, we characterized CpxR phosphorylation (CpxR∼P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR∼P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR∼P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate. Cpx pa...

Virus research, 2011

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. I... more The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in s...

Traffic, 2004

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, cond... more The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleocytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

The Journal of Cell Biology, 2009

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and bi... more The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

Proceedings of the National Academy of Sciences, 2003

The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have b... more The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions. This paper was submitted directly (Track II) to the PNAS office.

Plant and Cell Physiology, 2009

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins ... more Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

Photosynthesis Research, 2008

The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similari... more The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of proteins belonging to the CAB family of light-harvesting complex proteins in plants. The SCP proteins are transiently expressed at high light intensity and other stress conditions but their exact function remains largely unknown. Recently we showed association of ScpD with light-stressed, monomeric Photosystem II in Synechocystis sp. PCC 6803 (Yao et al. J Biol Chem 282:267-276, 2007). Here we show that ScpB associates with Photosystem II at normal growth conditions. Moreover, upon introduction of a construct into Synechocystis so that ScpB is expressed continuously under normal growth conditions, ScpE was detected under non-stressed conditions as well, and was copurified with tagged ScpB and Photosystem II. We also report on a one-helix protein, Slr1544, that is somewhat similar to the SCPs and whose gene is cotranscribed with that of ScpD; Slr1544 is another member of the extended light-harvesting-like (Lil) protein family, and we propose to name it LilA.

Molecular & Cellular Proteomics, 2004

In this report, we describe a newly developed method for isolating outer membranes from Synechocy... more In this report, we describe a newly developed method for isolating outer membranes from Synechocystis sp. PCC 6803 cells. The purity of the outer membrane fraction was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. We investigated the protein composition of the outer membrane using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by database identification. Forty-nine proteins were identified corresponding to 29 different gene products. All of the identified proteins have a putative N-terminal signal peptide. About 40% of the proteins identified represent hypothetical proteins with unknown function. Among the proteins identified are a Toc75 homologue, a protein that was initially found in the outer envelope of chloroplasts in pea, as well as TolC, putative porins, and a pilus protein. Other proteins identified include ABC transporters and GumB, which has a suggested function in carbohydrate export. A number of proteases such as HtrA were also found in the outer membrane of Synechocystis sp. PCC 6803.

Journal of Proteomics, 2011

It is an established fact that allelic variation and post-translational modifications create diff... more It is an established fact that allelic variation and post-translational modifications create different variants of proteins, which are observed as isoelectric and size subspecies in two-dimensional gel based proteomics. Here we explore the stromal proteome of spinach and Arabidopsis chloroplast and show that clustering of mass spectra is a useful tool for investigating such variants and detecting modified peptides with amino acid substitutions or post-translational modifications.

Journal of Biological Chemistry, 2002

In plants, the chloroplast organelles host the photosynthetic machinery, which catalyzes the conv... more In plants, the chloroplast organelles host the photosynthetic machinery, which catalyzes the conversion of light energy to chemical energy used for synthesis of carbohydrates. Inside the chloroplast, the lumen compartment forms an integral part of the thylakoid network that performs the light reactions of photosynthesis. Despite intensive research within the field of photosynthesis, the lumen located proteins were relatively unexplored. To get insight into the lumen proteins and their roles in photosynthesis this thesis aimed at characterising the chloroplast lumen proteome.

Journal of Biological Chemistry, 2007

The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble tra... more The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.

FEBS Letters, 2000

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidops... more A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.

FEBS Letters, 2002

Spermatid-speci¢c thioredoxin-1 (Sptrx-1) is the ¢rst member of the thioredoxin family of protein... more Spermatid-speci¢c thioredoxin-1 (Sptrx-1) is the ¢rst member of the thioredoxin family of proteins with a tissue-spe-ci¢c expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel ¢ltration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disul¢de bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1^360 and 361^469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disul¢de cross-linking) of the di¡erent structures in the developing tail of spermatids and spermatozoa. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

BMC Musculoskeletal Disorders, 2011

The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated w... more The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis.

The Plant Journal, 2006

† The first two authors contributed equally to this paper, and this work is the result of an equa... more † The first two authors contributed equally to this paper, and this work is the result of an equal collaboration between the laboratories of the last two authors.

Photosynthesis Research, 2003

Recent research in proteomics of the higher plant chloroplast has achieved considerable progress ... more Recent research in proteomics of the higher plant chloroplast has achieved considerable progress and added to our knowledge of lumenal chloroplast proteins. This work shows that chloroplast lumen has its own specific proteome and may comprise as many as 80 proteins. Although the new map of the lumenal proteome provides a great deal of information, it also raises numerous questions because the physiological functions of most of the novel lumenal proteins are unknown. In this Minireview, we summarize the latest discoveries regarding lumenal proteins and present the currently known facts about the lumenal chloroplast proteome of higher plants.

Photosynthesis Research, 2003

Currently, relatively few proteomics studies of chloroplast have been published, but the field ha... more Currently, relatively few proteomics studies of chloroplast have been published, but the field has just started emerging and is likely to develop more rapidly in the future. While the complex membrane structure of the chloroplast makes it difficult to study its entire proteome by global approaches, proteomics has considerably increased our knowledge of the proteins of single compartments such as, for instance, the envelope and the thylakoid lumen. Proteomics has also succeeded in the subunit characterisation of select protein complexes such as the ribosomes and the cytochrome b 6 f complex. In addition, proteomics was successfully applied to find new potential target pathways for thioredoxin-mediated signal transduction. In this review, we present an overview of the latest developments in the field of chloroplast proteomics and discuss their impact on photosynthesis research. In addition, we summarise the current state of research in proteomics of the photosynthetic cyanobactrium Synechocystis sp. PCC 6803.

PloS one, 2015

Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive... more Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. Outer membrane vesicles (OMVs) released by this species have been demonstrated to deliver effector proteins such as cytolethal distending toxin (CDT) and leukotoxin (LtxA) into human host cells and to act as triggers of innate immunity upon carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs). To improve our understanding of the pathogenicity-associated functions that A. actinomycetemcomitans exports via OMVs, we studied the proteome of density gradient-purified OMVs from a rough-colony type clinical isolate, strain 173 (serotype e) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This analysis yielded the identification of 151 proteins, which were found in at least three out of four independent experiments. Data are available via ProteomeXchange with identifier PXD002509. Through this study, we no...

PloS one, 2011

RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels... more RovA is a global transcriptional regulator of gene expression in pathogenic Yersinia. RovA levels are kept in check by a sophisticated layering of distinct transcriptional and post-transcriptional regulatory mechanisms. In the enteropathogen Y. pseudotuberculosis, we have previously reported that the extracytoplasmic stress sensing CpxA-CpxR two-component regulatory system modulates rovA expression. In this study, we characterized CpxR phosphorylation (CpxR∼P) in vitro, and determined that phosphorylation was necessary for CpxR to efficiently bind to the PCR-amplified upstream regulatory region of rovA. The precise CpxR∼P binding site was mapped by a nuclease protection assay and directed mutagenesis confirmed that in vivo binding to the rovA promoter inhibits transcription. Reduced RovA production was most pronounced following CpxR∼P accumulation in the Yersinia cytoplasm during chronic Cpx pathway activation and by the indiscriminate phosphodonor action of acetyl phosphate. Cpx pa...

Virus research, 2011

The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. I... more The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in s...

Traffic, 2004

The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, cond... more The nuclear pore complexes (NPCs), multiprotein assemblies embedded in the nuclear envelope, conduct nucleocytoplasmic traffic of macromolecules. Mimics of NPCs, called annulate lamellae pore complexes (ALPCs), are usually found in cytoplasmic membranous stacks in oocytes and early embryonic cells. They are believed to constitute storage compartments for excess premade nucleoporins. To evaluate the extent to which ALPCs store nucleoporins in early embryonic cells we took advantage of syncytial Drosophila embryos, containing both AL and rapidly proliferating nuclei in the common cytoplasm. Electron microscopic morphometric analysis showed that the number of ALPCs did not decrease to compensate for the growing number of NPCs during syncytial development. We performed Western blot analysis to quantify seven different nucleoporins and analyzed their intraembryonal distribution by confocal microscopy and subcellular fractionation. Syncytial embryos contained a large maternally contributed stockpile of nucleoporins. However, even during interphases, only a small fraction of the excess nucleoporins was assembled into ALPCs, whereas the major fraction was soluble and contained at least one phosphorylated nucleoporin. We conclude that in Drosophila embryos ALPCs play only a minor role in storing the excess maternally contributed nucleoporins. Factors that may prevent nucleoporins from assembly into ALPCs are discussed.

The Journal of Cell Biology, 2009

The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and bi... more The nuclear pore complex (NPC) mediates all nucleocytoplasmic transport, yet its structure and biogenesis remain poorly understood. In this study, we have functionally characterized interaction partners of the yeast transmembrane nucleoporin Ndc1. Ndc1 forms a distinct complex with the transmembrane proteins Pom152 and Pom34 and two alternative complexes with the soluble nucleoporins Nup53 and Nup59, which in turn bind to Nup170 and Nup157. The transmembrane and soluble Ndc1-binding partners have redundant functions at the NPC, and disruption of both groups of interactions causes defects in Ndc1 targeting and in NPC structure accompanied by significant pore dilation. Using photoconvertible fluorescent protein fusions, we further show that the depletion of Pom34 in cells that lack NUP53 and NUP59 blocks new NPC assembly and leads to the reversible accumulation of newly made nucleoporins in cytoplasmic foci. Therefore, Ndc1 together with its interaction partners are collectively essential for the biosynthesis and structural integrity of yeast NPCs.

Proceedings of the National Academy of Sciences, 2003

The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have b... more The characteristics of a phosphoprotein with a relative electrophoretic mobility of 12 kDa have been unknown during two decades of studies on redox-dependent protein phosphorylation in plant photosynthetic membranes. Digestion of this protein from spinach thylakoid membranes with trypsin and subsequent tandem nanospray-quadrupole-time-of-flight mass spectrometry of the peptides revealed a protein sequence that did not correspond to any previously known protein. Sequencing of the corresponding cDNA uncovered a gene for a precursor protein with a transit peptide followed by a strongly basic mature protein with a molecular mass of 8,640 Da. Genes encoding homologous proteins were found on chromosome 3 of Arabidopsis and rice as well as in ESTs from 20 different plant species, but not from any other organisms. The protein can be released from the membrane with high salt and is also partially released in response to light-induced phosphorylation of thylakoids, in contrast to all other known thylakoid phosphoproteins, which are integral to the membrane. On the basis of its properties, this plant-specific protein is named thylakoid soluble phosphoprotein of 9 kDa (TSP9). Mass spectrometric analyses revealed the existence of non-, mono-, di-, and triphosphorylated forms of TSP9 and phosphorylation of three distinct threonine residues in the central part of the protein. The phosphorylation and release of TSP9 from the photosynthetic membrane on illumination favor participation of this basic protein in cell signaling and regulation of plant gene expression in response to changing light conditions. This paper was submitted directly (Track II) to the PNAS office.

Plant and Cell Physiology, 2009

Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins ... more Chloroplast thylakoid lumen of Arabidopsis thaliana contains 16 immunophilins, five cyclophilins and 11 FK506-binding proteins (FKBPs), which are considered protein folding catalysts, although only two of them, AtFKBP13 and AtCYP20-2, possess peptidyl-prolyl cis/trans isomerase (PPIase) activity. To address the question of the physiological significance of this activity, we obtained and characterized Arabidopsis mutants deficient in the most active PPIase, AtFKBP13, and a double mutant deficient in both AtFKBP13 and AtCYP20-2. Two-dimensional gel electrophoresis of isolated thylakoid lumen, as well as immunoblotting analyses of major photosynthetic membrane protein complexes did not reveal differences in protein composition between the mutants and the wild type. No changes in the relative content of photosynthetic proteins were found by differential stable isotope labeling and liquid chromatography-mass spectrometry (LC-MS) analyses. PPIase activity was measured in vitro in isolated thylakoid lumen samples using two different synthetic peptide substrates. Depending on the peptide substrate used for the assay, the PPIase activity in the thylakoid lumen of the mutants lacking either AtFKBP13 or both AtFKBP13 and AtCYP20-2 was as low as 10 or 2% of that in the wild type. Residual PPIase activity detected in the double mutant originated from AtCYP20-3, a cyclophilin from chloroplast stroma contaminating thylakoid lumen preparations. None of the mutants differed from the wild-type plants when grown under normal, cold stress or high light conditions. It is concluded that cellular functions of immunophilins in the thylakoid lumen of chloroplasts are not related to their PPIase capacity and should be investigated beyond this enzymatic activity.

Photosynthesis Research, 2008

The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similari... more The cyanobacterial small CAB-like proteins (SCPs) are one-helix proteins with compelling similarity to the first and third transmembrane helix of proteins belonging to the CAB family of light-harvesting complex proteins in plants. The SCP proteins are transiently expressed at high light intensity and other stress conditions but their exact function remains largely unknown. Recently we showed association of ScpD with light-stressed, monomeric Photosystem II in Synechocystis sp. PCC 6803 (Yao et al. J Biol Chem 282:267-276, 2007). Here we show that ScpB associates with Photosystem II at normal growth conditions. Moreover, upon introduction of a construct into Synechocystis so that ScpB is expressed continuously under normal growth conditions, ScpE was detected under non-stressed conditions as well, and was copurified with tagged ScpB and Photosystem II. We also report on a one-helix protein, Slr1544, that is somewhat similar to the SCPs and whose gene is cotranscribed with that of ScpD; Slr1544 is another member of the extended light-harvesting-like (Lil) protein family, and we propose to name it LilA.

Molecular & Cellular Proteomics, 2004

In this report, we describe a newly developed method for isolating outer membranes from Synechocy... more In this report, we describe a newly developed method for isolating outer membranes from Synechocystis sp. PCC 6803 cells. The purity of the outer membrane fraction was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. We investigated the protein composition of the outer membrane using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by database identification. Forty-nine proteins were identified corresponding to 29 different gene products. All of the identified proteins have a putative N-terminal signal peptide. About 40% of the proteins identified represent hypothetical proteins with unknown function. Among the proteins identified are a Toc75 homologue, a protein that was initially found in the outer envelope of chloroplasts in pea, as well as TolC, putative porins, and a pilus protein. Other proteins identified include ABC transporters and GumB, which has a suggested function in carbohydrate export. A number of proteases such as HtrA were also found in the outer membrane of Synechocystis sp. PCC 6803.

Journal of Proteomics, 2011

It is an established fact that allelic variation and post-translational modifications create diff... more It is an established fact that allelic variation and post-translational modifications create different variants of proteins, which are observed as isoelectric and size subspecies in two-dimensional gel based proteomics. Here we explore the stromal proteome of spinach and Arabidopsis chloroplast and show that clustering of mass spectra is a useful tool for investigating such variants and detecting modified peptides with amino acid substitutions or post-translational modifications.

Journal of Biological Chemistry, 2002

In plants, the chloroplast organelles host the photosynthetic machinery, which catalyzes the conv... more In plants, the chloroplast organelles host the photosynthetic machinery, which catalyzes the conversion of light energy to chemical energy used for synthesis of carbohydrates. Inside the chloroplast, the lumen compartment forms an integral part of the thylakoid network that performs the light reactions of photosynthesis. Despite intensive research within the field of photosynthesis, the lumen located proteins were relatively unexplored. To get insight into the lumen proteins and their roles in photosynthesis this thesis aimed at characterising the chloroplast lumen proteome.

Journal of Biological Chemistry, 2007

The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble tra... more The cyanobacterial small CAB-like proteins (SCPs) consist of one-helix proteins that resemble transmembrane regions of the light-harvesting proteins of plants. To determine whether these proteins are associated with protein complexes in the thylakoid membrane, an abundant member of the SCP family, ScpD, was marked with a His tag, and proteins co-isolating with His-tagged ScpD were identified. These proteins included the major Photosystem (PS) II components as well as FtsH, which is involved in degradation of the PSII complex. To ascertain specific interaction between ScpD and the PSII complex, the His-tagged protein fraction was subjected to two-dimensional blue native/SDS-PAGE. Again, PSII components were co-isolated with ScpD-His, and ScpD-His was found to interact most strongly with CP47. ScpD association was most prominent with the monomeric form of PSII, suggesting ScpD association with PSII that is repaired. Using antibodies that recognize both ScpC and ScpD, we found the ScpC protein, which is very similar in primary structure to ScpD, to also co-isolate with the PSII complex. In contrast, ScpE did not co-isolate with a major protein complex in thylakoids. A fourth member of the SCP family, ScpB, could not be immunodetected, but was found by mass spectrometry in samples co-isolating with ScpD-His. Therefore, ScpB may be associated with ScpD as well. No association between SCPs and PSI could be demonstrated. On the basis of these and other data presented, we suggest that members of the SCP family can associate with damaged PSII and can serve as a temporary pigment reservoir while PSII components are being replaced.

FEBS Letters, 2000

A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidops... more A study by two-dimensional electrophoresis showed that the soluble, lumenal fraction of Arabidopsis thaliana thylakoids can be resolved into 300 protein spots. After subtraction of low-intensity spots and accounting for low-level stromal contamination, the number of more abundant, lumenal proteins was estimated to be between 30 and 60. Two of these proteins have been identified: a novel plastocyanin that also was the predominant component of the total plastocyanin pool, and a putative ascorbate peroxidase. Import studies showed that these proteins are routed to the thylakoid lumen by the Sec- and delta pH-dependent translocation pathways, respectively. In addition, novel isoforms of PsbO and PsbQ were identified.

FEBS Letters, 2002

Spermatid-speci¢c thioredoxin-1 (Sptrx-1) is the ¢rst member of the thioredoxin family of protein... more Spermatid-speci¢c thioredoxin-1 (Sptrx-1) is the ¢rst member of the thioredoxin family of proteins with a tissue-spe-ci¢c expression pattern, found exclusively in the tail of elongating spermatids and spermatozoa. We describe here further biochemical characterization of human Sptrx-1 protein structure and enzymatic activity. In gel ¢ltration chromatography human Sptrx-1 eluates as a 400 kDa protein consistent with either an oligomeric form, not maintained by intermolecular disul¢de bonding, and/or a highly asymmetrical structure. Analysis of circular dichroism spectra of fragments 1^360 and 361^469 and comparison to spectra of full-length Sptrx-1 supports a two-domain organization with a largely unstructured N-terminal domain and a folded thioredoxin-like C-terminal domain. Functionally, Sptrx-1 behaves as an oxidant in vitro when using selenite, but not oxidized glutathione, as electron acceptor. This oxidizing enzymatic activity suggests that Sptrx-1 might govern the stabilization (by disul¢de cross-linking) of the di¡erent structures in the developing tail of spermatids and spermatozoa. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

BMC Musculoskeletal Disorders, 2011

The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated w... more The trapezius muscle is a neck muscle that is susceptible to chronic pain conditions associated with repetitive tasks, commonly referred to as chronic work-related myalgia, hence making the trapezius a muscle of clinical interest. To provide a basis for further investigations of the proteomic traits of the trapezius muscle in disease, two-dimensional difference gel electrophoresis (2D-DIGE) was performed on the healthy trapezius using vastus lateralis as a reference. To obtain as much information as possible from the vast proteomic data set, both one-way ANOVA, with and without false discovery rate (FDR) correlation, and partial least square projection to latent structures with discriminant analysis (PLS-DA) were combined to compare the outcome of the analysis.

The Plant Journal, 2006

† The first two authors contributed equally to this paper, and this work is the result of an equa... more † The first two authors contributed equally to this paper, and this work is the result of an equal collaboration between the laboratories of the last two authors.