RESUMO
Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.
Assuntos
Proteínas de Ligação ao Cálcio/química , Cálcio/química , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/química , Tiorredoxinas/química , Sítios de Ligação , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Transporte de Elétrons , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Modelos Moleculares , Oxirredução , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Fotossíntese/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMO
The understanding of calcium as a second messenger in plants has been growing intensively over the last decades. Recently, attention has been drawn to the organelles, especially the chloroplast but focused on the stromal Ca2+ transients in response to environmental stresses. Herein we will expand this view and discuss the role of Ca2+ in photosynthesis. Moreover we address of how Ca2+ is delivered to chloroplast stroma and thylakoids. Thereby, new light is shed on the regulation of photosynthetic electron flow and light-dependent metabolism by the interplay of Ca2+, thylakoid acidification and redox status. This article is part of a Special Issue entitled: Chloroplast biogenesis.
Assuntos
Cálcio/fisiologia , Fotossíntese , Proteínas de Arabidopsis/fisiologia , Calmodulina/fisiologia , Cloroplastos/metabolismo , Transporte de Elétrons , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologiaRESUMO
Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-O-methylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of (18)O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants.
