ABSTRACT
A chloroplast protein disulfide isomerase (PDI) was previously proposed to regulate translation of the unicellular green alga Chlamydomonas reinhardtii chloroplast psbA mRNA, encoding the D1 protein, in response to light. Here we show that AtPDI6, one of 13 Arabidopsis thaliana PDI genes, also plays a role in the chloroplast. We found that AtPDI6 is targeted and localized to the chloroplast. Interestingly, AtPDI6 knockdown plants displayed higher resistance to photoinhibition than wild-type plants when exposed to a tenfold increase in light intensity. The AtPDI6 knockdown plants also displayed a higher rate of D1 synthesis under a similar light intensity. The increased resistance to photoinhibition may not be rationalized by changes in antenna or non-photochemical quenching. Thus, the increased D1 synthesis rate, which may result in a larger proportion of active D1 under light stress, may led to the decrease in photoinhibition. These results suggest that, although the D1 synthesis rates observed in wild-type plants under high light intensities are elevated, repair can potentially occur faster. The findings implicate AtPDI6 as an attenuator of D1 synthesis, modulating photoinhibition in a light-regulated manner.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Chloroplast Proteins/metabolism , Photosystem II Protein Complex/biosynthesis , Protein Disulfide-Isomerases/metabolism , Arabidopsis/genetics , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Chloroplast Proteins/genetics , Chloroplasts/genetics , Chloroplasts/metabolism , Chloroplasts/radiation effects , Gene Knockdown Techniques , Light Signal Transduction , Protein Disulfide-Isomerases/genetics , RNA, Messenger/metabolismSubject(s)
Hypnosis, Anesthetic/statistics & numerical data , Hypnosis/statistics & numerical data , Outcome Assessment, Health Care/statistics & numerical data , Smoking Cessation/statistics & numerical data , Bias , Follow-Up Studies , Humans , Hypnosis/methods , Surveys and Questionnaires , Treatment OutcomeABSTRACT
The reduction and the formation of regulatory disulfide bonds serve as a key signaling element in chloroplasts. Members of the thioredoxin (Trx) superfamily of oxidoreductases play a major role in these processes. We have characterized a small family of plant-specific Trxs in Arabidopsis (Arabidopsis thaliana) that are rich in cysteine and histidine residues and are typified by a variable noncanonical redox active site. We found that the redox midpoint potential of three selected family members is significantly less reducing than that of the classic Trxs. Assays of subcellular localization demonstrated that all proteins are localized to the chloroplast. Selected members showed high activity, contingent on a dithiol electron donor, toward the chloroplast 2-cysteine peroxiredoxin A and poor activity toward the chloroplast NADP-malate dehydrogenase. The expression profile of the family members suggests that they have distinct roles. The intermediate redox midpoint potential value of the atypical Trxs might imply adaptability to function in modulating the redox state of chloroplast proteins with regulatory disulfides.
Subject(s)
Chloroplast Thioredoxins/metabolism , Chloroplasts/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Chloroplast Thioredoxins/chemistry , Chloroplasts/enzymology , Cysteine/metabolism , Enzyme Activation , Gene Expression Regulation, Plant , Molecular Sequence Data , Oxidation-Reduction , Peroxiredoxins/metabolism , Phylogeny , Protein Transport , Sequence AlignmentABSTRACT
Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence. This novel domain architecture led to the hypothesis that MIN1 is in the plasma membrane or the chloroplast envelope, where membrane association of the C2 domain promotes proper eyespot organization. Mutation of conserved C2 domain loop residues disrupted association of the MIN1 C2 domain with the chloroplast envelope in moss cells but did not abolish eyespot assembly in Chlamydomonas. In min1 null cells, channelrhodopsin-1 (ChR1) photoreceptor levels were reduced, indicating a role for MIN1 in ChR1 expression and/or stability. However, ChR1 localization was only minimally disturbed during photoautotrophic growth of min1 cells, conditions under which the pigment granule layers are disorganized. The data are consistent with the hypothesis that neither MIN1 nor proper organization of the plastidic components of the eyespot is essential for localization of ChR1.
Subject(s)
Algal Proteins/chemistry , Chlamydomonas reinhardtii/chemistry , Chlamydomonas reinhardtii/physiology , Algal Proteins/genetics , Algal Proteins/metabolism , Amino Acid Sequence , Animals , Chlamydomonas reinhardtii/genetics , Chloroplasts/metabolism , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
RB60 is an atypical protein disulfide isomerase (PDI) that functions as a member of a redox regulatory protein complex controlling translation in the chloroplast of Chlamydomonas reinhardtii, but also contains a C-terminal endoplasmic reticulum (ER) retention signal, -KDEL. Here, we show by fluorescence microscopy that RB60 resides in the chloroplast but also outside of the chloroplast colocalized with BiP, an ER marker protein. RB60 accumulates in microsomes that exhibit a typical ER magnesium-shift, and cotranslationally translocates into ER microsomes. The first 50-aa leader of RB60 is sufficient for both chloroplast and ER targeting. The leader is cleaved upon translocation into the ER, whereas it remains intact after import to the chloroplast. The leader sequence also contains an acidic domain that appears necessary for the protein's association with the thylakoid membranes. Based on these and additional results, we propose that the dual localization of RB60 occurs via the two conserved transport mechanisms, to the chloroplast and to the ER, that the chloroplast RB60 most likely carries an additional function in the ER, and that its mode of transport, including the differential cleavage of its N terminus, plays an important role in its suborganellar localization and organellar-specific function.
Subject(s)
Chlamydomonas reinhardtii/enzymology , Chloroplasts/enzymology , Endoplasmic Reticulum/enzymology , Protein Disulfide-Isomerases/metabolism , Amino Acid Sequence , Animals , Chlamydomonas reinhardtii/growth & development , Humans , Molecular Sequence Data , Pisum sativum/enzymology , Protein Disulfide-Isomerases/chemistry , Protein Disulfide-Isomerases/genetics , Protein Disulfide-Isomerases/isolation & purification , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The yeast and human mitochondrial sulfhydryl oxidases of the Erv1/Alr family have been shown to be essential for the biogenesis of mitochondria and the cytosolic iron sulfur cluster assembly. In this study we identified a likely candidate for the first mitochondrial flavin-linked sulfhydryl oxidase of the Erv1-type from a photosynthetic organism. The central core of the plant enzyme (AtErv1) exhibits all of the characteristic features of the Erv1/Alr protein family, including a redox-active YPCXXC motif, noncovalently bound FAD, and sulfhydryl oxidase activity. Transient expression of fusion proteins of AtErv1 and the green fluorescence protein in plant protoplasts showed that the plant enzyme preferentially localizes to the mitochondria. Yet AtErv1 has several unique features, such as the presence of a CXXXXC motif in its carboxyl-terminal domain and the absence of an amino-terminally localized cysteine pair common to yeast and human Erv1/Alr proteins. In addition, the dimerization of AtErv1 is not mediated by its amino terminus but by its unique CXXXXC motif. In vitro assays with purified protein and artificial substrates demonstrate a preference of AtErv1 for dithiols with a defined space between the thiol groups, suggesting a thioredoxin-like substrate.