Importance of Post-Translational Modifications for Functionality of a Chloroplast-Localized Carbonic Anhydrase (CAH1) in Arabidopsis thaliana

Background: 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 non-canonical pathway is beneficial for certain chloroplast resident proteins is not yet known. Therefore, to elucidate the significance of glycosylation in trafficking and the effect of glycosylation on the stability and function of the protein, epitope-labelled wild type and mutated versions of CAH1 were expressed in plant cells. Methodology/Principal Findings: Transient expression of mutant CAH1 with disrupted glycosylation sites showed that the protein harbours four, or in certain cases five, N-glycans. While the wild type protein trafficked through the secretory pathway to the chloroplast, the non-glycosylated protein formed aggregates and associated with the ER chaperone BiP, indicating that glycosylation of CAH1 facilitates folding and ER-export. Using cysteine mutants we also assessed the role of disulphide bridge formation in the folding and stability of CAH1. We found that a disulphide bridge between cysteines at positions 27 and 191 in the mature protein was required for correct folding of the protein. Using a mass spectrometric approach we were able to measure the enzymatic activity of CAH1 protein. Under circumstances where protein N-glycosylation is blocked in vivo, the activity of CAH1 is completely inhibited. Conclusions/Significance: We show for the first time the importance of post-translational modifications such as N-glycosylation and intramolecular disulphide bridge formation in folding and trafficking of a protein from the secretory pathway to the chloroplast in higher plants. Requirements for these post-translational modifications for a fully functional native protein explain the need for an alternative route to the chloroplast.This work was supported by the Swedish Research Council (VR), the Kempe Foundations and Carl Tryggers Foundation to GS, and grant numbers BIO2006-08946 and BIO2009-11340 from the Spanish Ministerio de Ciencia e Innovación (MICINN) to A

Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources?

Biosensors based on luminescent bacteria may be valuable tools to monitor the chemical quality and safety of surface and drinking water. In this review, an overview is presented of the recombinant strains available that harbour the bacterial luciferase genes luxCDABE, and which may be used in an online biosensor for water quality monitoring. Many bacterial strains have been described for the detection of a broad range of toxicity parameters, including DNA damage, protein damage, membrane damage, oxidative stress, organic pollutants, and heavy metals. Most lux strains have sensitivities with detection limits ranging from milligrams per litre to micrograms per litre, usually with higher sensitivities in compound-specific strains. Although the sensitivity of lux strains can be enhanced by various molecular manipulations, most reported detection thresholds are still too high to detect levels of individual contaminants as they occur nowadays in European drinking waters. However, lux strains sensing specific toxic effects have the advantage of being able to respond to mixtures of contaminants inducing the same effect, and thus could be used as a sensor for the sum effect, including the effect of compounds that are as yet not identified by chemical analysis. An evaluation of the suitability of lux strains for monitoring surface and drinking water is therefore provided

Salt pans as a new archaeological sea-level proxy: A test case from Dalmatia, Croatia

Reconstruction of paleo relative sea level (RSL) is based on multi-proxy disciplines including archaeology. Saltpans, like fish tanks which are considered a reliable method for acquiring RSL index points, are also anthropogenic intertidal facilities, used continuously in the Mediterranean from early antiquity. The Dalmatian shore contains a large number of preserved and historically dated ancient saltpans, now flooded by the rising sea, providing great potential for past RSL indication. The primary objective of this study is to develop a new holistic approach for producing high quality elevation measurements of the saltpan structures and estimating paleo RSL during the last 2 ka. The study combined aerial photo- grammetry of the site, bathymetry acoustics scanning, and underwater archaeological survey of the man- made structures, as well as sampling wood and mortar in situ where available. Evaluation of each site\u2019s RSL and the functional height is based on the same assumptions and interpretations made for fish tanks, using elevation measurements on the top of the separation walls and the bottom of the sluice gates. In all the sites analyzed here, we achieved reliable digital surface models with continuous high- resolution data on the indicative structures with single centimeters level of accuracy. The study finds that during the 5th - 6th centuries, RSL was 92 \ub1 25 cm, increased to 62 \ub1 21 cm during the 7th - 11th centuries, and decreased to 104 \ub1 20 cm in the 14th century. Medieval RSL can be explained either by strong tectonic subsidence post 1300 AD or by fluctuations as observed in the East Mediterranean, which requires validation by further Medieval indicators