RESUMEN
The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.
Asunto(s)
Chlamydomonas/metabolismo , Proteínas de la Membrana/metabolismo , Fotosíntesis , Proteínas de Plantas/metabolismo , Tilacoides/metabolismo , Chlamydomonas/genética , Chlamydomonas/efectos de la radiación , Regulación de la Expresión Génica de las Plantas , Luz , Proteínas de la Membrana/genética , Mutación , Complejo de Proteína del Fotosistema II/metabolismo , Proteínas de Plantas/genética , Proteómica , Interferencia de ARN , Tilacoides/ultraestructuraRESUMEN
Knowledge of the interaction partners of a protein of interest may provide important information on its function. Common to currently available tools for the identification of protein-protein interactions, however, is their high rates of false positives. Only recently an assay was reported that allowed for the unequivocal identification of protein-protein interactions in mammalian cells in a single experiment. This assay, termed quantitative immunoprecipitation combined with knockdown (QUICK), combines RNAi, stable isotope labeling with amino acids in cell culture, immunoprecipitation, and quantitative MS. We are using the unicellular green alga Chlamydomonas reinhardtii to understand the roles of chaperones in chloroplast biogenesis. The goal of this work was to apply QUICK to Chlamydomonas for the identification of novel interaction partners of vesicle-inducing protein in plastids 1 (VIPP1), a protein required for the biosynthesis/maintenance of thylakoid membranes and known substrate of chloroplast HSP70B. We report here a robust QUICK protocol for Chlamydomonas that has been improved (i) by introducing a cross-linking step (-X) to improve protein complex stability and (ii) by including a control for the correction of unequal immunoprecipitation and/or labeling efficiencies. Using QUICK and cross-linking we could verify that HSP70B and CGE1 form a complex with VIPP1 and could also demonstrate that chloroplast HSP90C is part of this complex. Moreover, we could show that the chaperones interact with VIPP1 also in membrane fractions.
Asunto(s)
Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Técnicas de Silenciamiento del Gen/métodos , Proteínas HSP90 de Choque Térmico/metabolismo , Inmunoprecipitación/métodos , Mapeo de Interacción de Proteínas/métodos , Animales , Chlamydomonas reinhardtii/química , Cloroplastos/química , Proteínas HSP70 de Choque Térmico/metabolismo , Marcaje Isotópico , Espectrometría de Masas/métodos , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/análisis , Proteínas de Plantas/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Tripsina/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Unión al Calcio/química , Calcio/química , Chlamydomonas reinhardtii/genética , Cloroplastos/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/química , Tiorredoxinas/química , Sitios de Unión , Calcio/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Calmodulina/química , Calmodulina/genética , Calmodulina/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Transporte de Electrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Modelos Moleculares , Oxidación-Reducción , Peroxirredoxinas/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Fotosíntesis/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC) and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants. Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for (15)N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE1.
Asunto(s)
Inmunoprecipitación/métodos , Espectrometría de Masas/métodos , Isótopos de Nitrógeno/análisis , Proteínas/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Chlamydomonas reinhardtii , Factores de Intercambio de Guanina Nucleótido/química , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Marcaje Isotópico/métodos , Isótopos de Nitrógeno/química , Proteínas de Plantas , Mapas de Interacción de Proteínas , Proteínas/química , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
The unicellular green alga Chlamydomonas reinhardtii has been used as a model organism for many decades, mainly to study photosynthesis and flagella/cilia. Only recently, Chlamydomonas has received much attention because of its ability to produce hydrogen and nonpolar lipids that have promise as biofuels. The best-studied multicellular cousin of Chlamydomonas reinhardtii is Volvox carteri, whose life cycle comprises events that have clear parallels in higher plants and/or animals, making it an excellent system in which to study fundamental developmental processes. Molecular chaperones are proteins that guide other cellular proteins through their life cycle. They assist in de novo folding of nascent chains, mediate assembly and disassembly of protein complexes, facilitate protein transport across membranes, disassemble protein aggregates, fold denatured proteins back to the native state, and transfer unfoldable proteins to proteolytic degradation. Hence, molecular chaperones regulate protein function under all growth conditions and play important roles in many basic cellular and developmental processes. The aim of this chapter is to describe recent advances toward understanding molecular chaperone biology in Chlamydomonas and Volvox.