Figure 1 - uploaded by Dmitry Shevela
Content may be subject to copyright.
Schematic view and amino acid sequence of the HA epitope-tagged CAH1 variants. (A) The five potential N-glycosylation sites and the four cysteine residues are marked by ''N'' and ''C'', respectively, and numbered according to their position in the mature native CAH1. SP, ER signal peptide; HA, HA-epitope tag; dCAH1, mature CAH1. (B) Position and sequence of the HA-tag is indicated together with the mutated cysteine residues (green) and N-glycosylation sites (red). HA epitope sequence is enlarged. doi:10.1371/journal.pone.0021021.g001
Source publication
The Arabidopsis CAH1 alpha-type carbonic anhydrase is one of the few plant proteins known to be targeted to the chloroplast through the secretory pathway. CAH1 is post-translationally modified at several residues by the attachment of N-glycans, resulting in a mature protein harbouring complex-type glycans. The reason of why trafficking through this...
Contexts in source publication
Context 1
... mature protein has five potential N-glycosylation sites and contains four cysteine residues. To study the function of these groups several mutant variants of the protein were generated, based on a Hemagglutinin (HA) epitope-tagged version of CAH1 ( Figure 1, Table 1, Table S1). The HA-tagged CAH1 (HC) was stably transformed into an Arabidopsis thaliana cell suspension culture and sub-cellular localization of the expressed protein was analyzed using immunogold (IG) labelling followed by electron microscopy. ...
Context 2
... HA-tagged CAH1 (HC) was stably transformed into an Arabidopsis thaliana cell suspension culture and sub-cellular localization of the expressed protein was analyzed using immunogold (IG) labelling followed by electron microscopy. The wild type HC was mainly localized to the chloroplast of cells, where the highest immunogold labelling density was observed (Table 2, Figure S1). As evident from Table 2, some labelling associated with the ER and Golgi apparatus was also detected. ...
Context 3
... disulphide bridges are important for folding and ER-export of the CAH1 protein Mature CAH1 has four cysteines potentially involved in the formation of intra and/or intermolecular disulphide bridges ( Figure 1). The modelled 3D structure of CAH1 protein indicates that Cys27 and Cys191 are located in close proximity to each other ( Figure 6a). ...
Context 4
... identify cysteine residues involved in intramolecular bond formation, each of the four cysteine residues were individually mutated (C1-C4; Table 1, Figure 1). Under reducing conditions, each mutant protein migrated with a similar pattern to wt HC ( Figure 6b, lower panel). ...
Context 5
... concluded in Figure 2 and Figure S2, incomplete glycosylation of HC, specifically from partial occupation of site NVS194, results in two major glycoforms of the protein. Since only two residues separate Cys191 from NVS194 (Figure 1b), and because Cys27 and Cys191 appear to form a disulphide bridge, the C3 mutation could promote accumulation of the protein isoform harbouring five glycans by making NVS194 more accessible to the glycosylation machinery. This could explain the prevalence of the higher mass isoform of C3 seen in Figure 6b. ...
Context 6
... the biological significance of possession of a set of chloroplast glycoproteins targeted through the endomembrane system and explore the relationships between the structure, function and novel trafficking route to the chloroplast of CAH1, we expressed an epitope-tagged version of the wt protein (HC). Several mutant versions of the HC protein were also generated and transiently expressed in protoplasts to evaluate the role of the attached carbohydrate structures and its redox status ( Figure 1, Table 1, Table S1). Our data clearly showed that the epitope-tagged CAH1 protein was targeted to the chloroplast in Arabidopsis suspension culture cells. ...
Context 7
... previous study of the CAH1 protein showed that the protein is N-glycosylated, but the numbers and positions of the glycans in vivo were not resolved [7], therefore mutants to study glycosylation status were prepared (Figure 1, Table 1, Table S1). The electrophoretic migration pattern of the NG mutant protein was identical to that of HC expressed in the presence of the N- glycosylation inhibitor tunicamycin (Figure 2a), confirming that no additional sites other than the five mutated could harbour N- glycans in the CAH1 protein. ...
Citations
... Studies on Chlamydomonas have shown that TM and BFA treatment causes heightened sensitivity in cells lacking IRE1, a key sensor of ER stress [30]. Specifically, TM inhibits the N-glycosylation of glycoproteins, a process that is absent in chloroplasts [32,33]. This inhibition leads to the accumulation of misfolded proteins in the ER [34]. ...
Stress on the Endoplasmic reticulum (ER) can severely disrupt cellular function by impairing protein folding and post-translational modifications, thereby leading to the accumulation of poor-quality proteins. However, research on its impact on photosynthesis remains limited. In this study, we investigated the impact of ER stress on the photosynthetic efficiency of Chlamydomonas reinhardtii using pharmacological inducers, tunicamycin (TM) and brefeldin A (BFA), which specifically target the ER. Our measurements of photosynthetic parameters showed that these ER stress-inducing compounds caused a significant decline in photosynthetic efficiency. A proteomic analysis confirmed that TM and BFA effectively induce ER stress, as evidenced by the upregulation of ER stress-related proteins. Furthermore, we observed a widespread downregulation of photosynthesis-related proteins, which is consistent with the results obtained from our measurements of photosynthetic parameters. These findings suggest that the stress on ER has a profound impact on chloroplast function, disrupting photosynthetic processes. This study highlights the critical interdependence between the ER and chloroplasts, and it underscores the broader implications of ER stress on the cellular metabolism and energy efficiency of photosynthetic organisms.
... A large number of plant genes encode CA. In Arabidopsis thaliana, there are nineteen distinct genes in total encoding all α, β, and γ isoforms, including six AtαCAs, eight AtβCAs, three AtγCAs, and two AtγCALs (γ-like CAs) (Burén et al. 2011;Fabre et al. 2007). A similar number of CA genes present in the genomes of other monocots and dicots plants, such as nineteen, sixteen, and seventeen CA genes in Medicago truncatula, Oryza sativa, and Sorghum bicolor, respectively (DiMario et al. 2017). ...
Carbonic anhydrases (CAs), as zinc metalloenzymes, are ubiquitous in nature and play essential roles in diverse biological processes. Although CAs have been broadly explored and studied, comprehensive characteristics of CA gene family members in the soybean (Glycine max) are still lacking. A total of 35 CA genes (GmCAs) were identified; they distributed on sixteen chromosomes of the soybean genome and can be divided into three subfamilies (α-type, β-type, and γ-type). Bioinformatics analysis showed that the specific GmCA gene subfamily or clade exhibited similar characteristics and that segmental duplications took the major role in generating new GmCAs. Furthermore, the synteny and evolutionary constraints analyses of CAs among soybean and distinct species provided more detailed evidence for GmCA gene family evolution. Cis-element analysis of promoter indicated that GmCAs may be responsive to abiotic stress and regulate photosynthesis. Moreover, the expression patterns of GmCAs varied in different tissues at diverse developmental stages in soybean. Additionally, we found that eight representative GmCAs may be involved in the response of soybean to low phosphorus stress. The systematic investigation of the GmCA gene family in this study will provide a valuable basis for further functional research on soybean CA genes.
... The largest number of studies have been carried out on Arabidopsis thaliana, as this plant is widely used as a model organism. Within its genome eight genes encoding α-CAs have been revealed; among these, four have been functionally investigated and one underwent a partial biochemical characterization [49]. Clear information on the intracellular location of these α-CA isoforms is available only for α-CA1, which is located in chloroplast stroma [46], and α-CA4 and α-CA5 found in thylakoid membranes [50][51][52], whereas recent studies provided evidence of the presence of α-CA2 in thylakoid membranes too [53][54][55]. ...
... It contains several glycosylation sites that must be occupied by N-glycans for correct folding, trafficking, and functionality of the protein. In addition, the protein must be stabilized by a disulfide bridge between the conserved Cys27 and Cys191 residues for folding and endoplasmic reticulum (ER)export [46,49]. ...
Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure–function relationship.
... They are then imported in a cotranslational manner to go to the Golgi complex, where they are glycosylated before reaching the target plastids. The mechanism underlying the import from the Golgi complex is still obscure [166,167]. ...
The problem with increasing the yield of recombinant proteins is resolvable using different approaches, including the transport of a target protein to cell compartments with a low protease activity. In the cell, protein targeting involves short-signal peptide sequences recognized by intracellular protein transport systems. The main systems of the protein transport across membranes of the endoplasmic reticulum and endosymbiotic organelles are reviewed here, as are the major types and structure of the signal sequences targeting proteins to the endoplasmic reticulum and its derivatives, to plastids, and to mitochondria. The role of protein targeting to certain cell organelles depending on specific features of recombinant proteins and the effect of this targeting on the protein yield are discussed, in addition to the main directions of the search for signal sequences based on their primary structure. This knowledge makes it possible not only to predict a protein localization in the cell but also to reveal the most efficient sequences with potential biotechnological utility.
... Three potential N-glycosylation sites were predicted in hCA XII by the NetNGlyc 1.0 program (Gupta and Brunak 2002), namely N28, N80 and N162; however, a detailed experimental study carried out by Hong and coworkers indicated the occurrence of N-linked glycosylation only at residues N28 and N80, which are present in the extracellular region of the protein (Hong et al. 2015) (Fig. 4). A careful analysis of the hCA XII catalytic domain structure highlighted that these amino acids occur on the molecular surface, far away from the corresponding active site, thus suggesting that glycosylation is not important for catalytic function, but it is probably important for correct protein folding, ER secretion and subcellular localization, as observed for the glycosyation of chloroplast CA I from Arabidopsis thaliana (Buren et al. 2011). In agreement with this observation, Hong and coworkers demonstrated a clear correlation between the occurrence of N-glycosylation in hCA XII and protein subcellular localization (Hong et al. 2015). ...
Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.
... When a periplasmic CA is present, as it is the case in C. reinhardtii cells with an active CCM, its activity dominates the exchange kinetics (Sültemeyer and Rinast, 1996). MIMS is so far the most reliable method for CA activity measurements in biological samples and is particularly suitable for in vivo measurements on algae (Dang et al., 2014;Benlloch et al., 2015;Tolleter et al., 2017), cyanobacteria (Whitehead et al., 2014), corals (Tansik et al., 2015), and plants (Peltier et al., 1995;Clausen et al., 2005;Bureń et al., 2011;Benlloch et al., 2015). In vitro, the use MIMS and H 13 CO 3 − has also allowed unraveling the existence of a light-induced CO 2 production by the PSII (Koroidov et al., 2014;Shevela et al., 2020). ...
Since the first great oxygenation event, photosynthetic microorganisms have continuously shaped the Earth’s atmosphere. Studying biological mechanisms involved in the interaction between microalgae and cyanobacteria with the Earth’s atmosphere requires the monitoring of gas exchange. Membrane inlet mass spectrometry (MIMS) has been developed in the early 1960s to study gas exchange mechanisms of photosynthetic cells. It has since played an important role in investigating various cellular processes that involve gaseous compounds (O2, CO2, NO, or H2) and in characterizing enzymatic activities in vitro or in vivo. With the development of affordable mass spectrometers, MIMS is gaining wide popularity and is now used by an increasing number of laboratories. However, it still requires an important theory and practical considerations to be used. Here, we provide a practical guide describing the current technical basis of a MIMS setup and the general principles of data processing. We further review how MIMS can be used to study various aspects of algal research and discuss how MIMS will be useful in addressing future scientific challenges.
... However, although all CA families contain zinc, they appear to have evolved independently [7,8]. The model plant Arabidopsis thaliana contains 19 CAs (8 aCAs, 6 bCAs, and 5 cCAs), among which only aCA1-3 are expressed in leaves [9]. aCA1 protein is located in the Golgi apparatus membrane and, with the exception of roots, the aCA1gene is expressed in all plant parts [3,10]. ...
... The induction of CA1 expression at low CO 2 concentrations has been found to promote the balance between CO 2 and HCO 3 -, thereby facilitating the diffusion of CO 2 across the cell membrane and thus providing inorganic carbon for photosynthesis [11]. Consistently, silencing of the aCA1 gene has been demonstrated to reduce photosynthetic activity and starch accumulation in Arabidopsis mutants [9]. aCA4 is located in the thylakoid membranes and affects the activity of the external light harvesting antenna complexes [12,13], whereas CA6 is mainly located around the pyrenoids and may be involved in the incorporation of CO 2 into bicarbonate, thereby increasing the concentration of HCO 3 in the cytoplasmic matrix and thus ensuring the retention of inorganic carbon within the chloroplasts [14]. ...
... Early studies on Arabidopsis CAs demonstrated that the induction of CA1 at low CO 2 concentrations can promote the balance between CO 2 and HCO 3 -, such that CO 2 on the cell surface can diffuse across the cell membrane and thereby provide inorganic carbon for photosynthesis [11]. Consistently, aca1 mutants have been found to exhibit reduced photosynthetic activity and starch accumulation [9]. The bCAs have been shown to maintain the concentration of CO 2 in the vicinity of RuBisCO, promote the diffusion of CO 2 through chloroplast membranes, rapidly remove HCO 3 and release CO 2 [6]. ...
Genes in the carbonic anhydrase (CA) family encode zinc metalloenzymes that catalyze the reversible interconversion of carbon dioxide and water to bicarbonate and protons. Although CAs play key roles in diverse biological processes involving carboxylation and decarboxylation, including photosynthesis and respiration, plant growth and response to stress, the characteristics of CA gene family members in tomato remain unclear. In this study, we performed an exhaustive search of the tomato genome and accordingly identified 14 CA genes that are unevenly distributed on the 12 tomato chromosomes. We examined in detail the structures, conserved motifs, phylogenetic relationships and duplications of these genes, and for functional characterization, also undertook RNA-seq analyses to assess the transcript levels of CA genes in various tissues and organs and at different developmental stages. Furthermore, we investigated the expression patterns of the CA genes in response to salt stress. We found that some family members exhibited tissue-specific expression, whereas others were more ubiquitously expressed. Our results will provide a valuable foundation for further studies on the CA genes in tomato and other plants in the Solanaceae family.
... CAH1, alpha-type carbonic anhydrase (EC 4.2.1.1), is one of the few plant proteins known to be targeted to the chloroplast, and which is essential for efficient photosynthesis (Samuelsson and Karlsson 2001, Burén et al. 2011, Kupriyanova et al. 2017. The mature CAH1 harboring multiple glycoforms of N-glycans (Burén et al. 2011) might be a good substrate to study the relation between N-glycosylation and photosynthesis. ...
... is one of the few plant proteins known to be targeted to the chloroplast, and which is essential for efficient photosynthesis (Samuelsson and Karlsson 2001, Burén et al. 2011, Kupriyanova et al. 2017. The mature CAH1 harboring multiple glycoforms of N-glycans (Burén et al. 2011) might be a good substrate to study the relation between N-glycosylation and photosynthesis. ...
... It is well documented that abnormal N-glycans in ER are detected by ERQC (ER-associated quality control compartment) system and misfolded proteins are removed via the ERAD (ER-associated degradation) pathway (Hong et al. 2009, Aebi 2013, Strasser 2016. Previous reports suggest that a chloroplast-located protein CAH1 becomes N-glycosylated before entering the chloroplast through the secretory pathway (Villarejo et al. 2005, Burén et al. 2011, indicating that N-glycosylation plays the role in regulation of secretory pathway for chloroplast-located proteins. The well N-glycosylated CAH1 is transported into the chloroplast through the secretory pathway in the WT plants and then participates in the process of photosynthesis. ...
N-glycosylation is one of the most important protein modifications in eukaryotes. It has been well established that N-glycosylation plays multiple roles in regulating stress tolerance of plants. However, the effects and mechanism of N-glycosylation on photosynthesis have not been well understood. In the present study, an obvious decrease in photosynthetic capacity and dry mass were detected in alg3-3 and cgl1-1, two typical mutants in N-glycosylation process. The maximal photochemical efficiency of PSII decreased significantly in cgl1-1. The values of effective quantum yield of PSII photochemistry, rate of photosynthetic electron transport through PSII, and photochemical quenching coefficient, which reflected the photochemical efficiency of plants, decreased as well, while the values of quantum yield of nonregulated energy dissipation of PSII showed obvious enhancement, the similar tendency was also observed in alg3-3. Furthermore, we found that N-glycosylation was also required to maintain the stability of a chloroplast-located protein CAH1, which was closely related to photosynthesis. These results suggest that N-glycosylation plays crucial roles in maintaining photosynthetic efficiency.
... Proteins are N-glycosylated in the endoplasmic reticulum (ER) via a system involving a large number of proteins (Ceriotti et al. 1998). However, it has been shown that certain chloroplast proteins such as α-type carbonic anhydrases and amylases are N-glycosylated (Burén et al. 2011;Chen et al. 2004;Faye and Daniell 2006;Lehtimäki et al. 2015). Moreover, α-carbonic anhydrases in Arabidopsis are one of the most abundant proteins in chloroplasts, suggesting that a large amount of foreign proteins can be transported to chloroplasts via this route. ...
Recently, plants have emerged as a lucrative alternative system for the production of recombinant proteins, as recombinant proteins produced in plants are safer and cheaper than those produced in bacteria and animal cell-based production systems. To obtain high yields in plants, recombinant proteins are produced in chloroplasts using different strategies. The first strategy is based on chloroplast transformation, followed by gene expression and translation in chloroplasts. This has proven to be a powerful approach for the production of proteins at high levels. The second approach is based on nuclear transformation, followed by post-translational import of proteins from the cytosol into chloroplasts. In the nuclear transformation approach, foreign genes are stably integrated into the nuclear genome or transiently expressed in the nucleus by non-integrating T-DNA. Although this approach also has great potential for protein production at high levels, it has not been thoroughly investigated. In this review, we focus on nuclear transformation-based protein expression and its subsequent sequestration in chloroplasts, and summarize the different strategies used for high-level production of recombinant proteins. We also discuss future directions for further improvements in protein production in chloroplasts through nuclear transformation-based gene expression.
... Indeed, amino acid sequence analysis showed that these four α-class GcCAHs possess several putative N-glycosylation sites, which are represented as NXS/T (N, asparagin; X, any amino acid residues except proline; S, serine; and T, threonine) (Supplemental Figure S3). Moreover, Arabidopsis CAH1 is known to be glycosylated, and N-glycosylation is critical for its enzymatic activity (Burén et al. 2011). We examined whether these four proteins are N-glycosylated. ...
Key message
Red alga, Gracilariopsis chorda, contains seven carbonic anhydrases that can be grouped into α-, β- and γ-classes.
Abstract
Carbonic anhydrases (CAHs) are metalloenzymes that catalyze the reversible hydration of CO2. These enzymes are present in all living organisms and play roles in various cellular processes, including photosynthesis. In this study, we identified seven CAH genes (GcCAHs) from the genome sequence of the red alga Gracilariopsis chorda and characterized them at the molecular, cellular and biochemical levels. Based on sequence analysis, these seven isoforms were categorized into four α-class, one β-class, and two γ-class isoforms. RNA sequencing revealed that of the seven CAHs isoforms, six genes were expressed in G. chorda in light at room temperature. In silico analysis revealed that these seven isoforms localized to multiple subcellular locations such as the ER, mitochondria and cytosol. When expressed as green fluorescent protein fusions in protoplasts of Arabidopsis thaliana leaf cells, these seven isoforms showed multiple localization patterns. The four α-class GcCAHs with an N-terminal hydrophobic leader sequence localized to the ER and two of them were further targeted to the vacuole. GcCAHβ1 with no noticeable signal sequence localized to the cytosol. The two γ-class GcCAHs also localized to the cytosol, despite the presence of a predicted presequence. Based on these results, we propose that the red alga G. chorda also employs multiple CAH isoforms for various cellular processes such as photosynthesis.
