ABSTRACT
The related proteins D1 and D2 together build up the photosystem II reaction center. Synthesis of D1 (PsbA) is highly regulated in all photosynthetic organisms. The mechanisms and specific protein factors involved in controlled expression of the psbA gene in higher plants are highly elusive. Here, we report on the identification of a chloroplast-located protein, HCF244 (for high chlorophyll fluorescence244), which is essentially required for translational initiation of the psbA messenger RNA in Arabidopsis (Arabidopsis thaliana). The factor is highly conserved between land plants, algae, and cyanobacteria. HCF244 was identified by coexpression analysis of HCF173, which encodes a protein that is also necessary for psbA translational initiation and in addition for stabilization of this messenger RNA. Phenotypic characterization of the mutants hcf244 and hcf173 suggests that the corresponding proteins operate cooperatively during psbA translation. Immunolocalization studies detected the majority of the two proteins at the thylakoid membrane. Both HCF244 and HCF173 are members of the atypical short-chain dehydrogenase/reductase superfamily, a modified group, which has lost enzyme activity but acquires new functions in the metabolism of the cell.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/genetics , Butyryl-CoA Dehydrogenase/metabolism , Eukaryotic Initiation Factors/metabolism , Peptide Chain Initiation, Translational , Amino Acid Sequence , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/genetics , Butyryl-CoA Dehydrogenase/chemistry , Centrifugation, Density Gradient , Eukaryotic Initiation Factors/chemistry , Gene Expression Regulation, Plant , Genes, Plant/genetics , Molecular Sequence Data , Mutation/genetics , Photosynthesis/genetics , Photosystem II Protein Complex/metabolism , Phylogeny , Protein Binding/genetics , Protein Structure, Tertiary , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Plant/metabolism , Spectrum Analysis , Thylakoids/metabolismABSTRACT
The major L-type voltage-gated calcium channels in heart consist of an α1C (Ca(V)1.2) subunit usually associated with an auxiliary ß subunit (Ca(V)ß2). In embryonic cardiomyocytes, both the complete and the cardiac myocyte-specific null mutant of Ca(V)ß2 resulted in reduction of L-type calcium currents by up to 75%, compromising heart function and causing defective remodeling of intra- and extra-embryonic blood vessels followed by embryonic death. Here we conditionally excised the Ca(V)ß2 gene (cacnb2) specifically in cardiac myocytes of adult mice (KO). Upon gene deletion, Ca(V)ß2 protein expression declined by >96% in isolated cardiac myocytes and by >74% in protein fractions from heart. These latter protein fractions include Ca(V)ß2 proteins expressed in cardiac fibroblasts. Surprisingly, mice did not show any obvious impairment, although cacnb2 excision was not compensated by expression of other Ca(V)ß proteins or changes of Ca(V)1.2 protein levels. Calcium currents were still dihydropyridine-sensitive, but current density at 0 mV was reduced by <29%. The voltage for half-maximal activation was slightly shifted to more depolarized potentials in KO cardiomyocytes when compared with control cells, but the difference was not significant. In summary, Ca(V)ß2 appears to be a much stronger modulator of L-type calcium currents in embryonic than in adult cardiomyocytes. Although essential for embryonic survival, Ca(V)ß2 down-regulation in cardiomyocytes is well tolerated by the adult mice.
Subject(s)
Calcium Channels, L-Type/biosynthesis , Gene Expression Regulation , Muscle Proteins/biosynthesis , Myocardium/enzymology , Myocytes, Cardiac/metabolism , Animals , Calcium Channels, L-Type/genetics , Embryo, Mammalian/enzymology , Embryo, Mammalian/pathology , Fibroblasts/enzymology , Fibroblasts/pathology , Mice , Mice, Knockout , Muscle Proteins/genetics , Myocardium/pathology , Myocytes, Cardiac/pathology , Organ Specificity/geneticsABSTRACT
By now, little is known on L-type calcium channel (LTCC) subunits expressed in mouse heart. We show that CaVbeta2 proteins are the major CaVbeta components of the LTCC in embryonic and adult mouse heart, but that in embryonic heart CaVbeta3 proteins are also detectable. At least two CaVbeta2 variants of approximately 68 and approximately 72 kDa are expressed. To identify the underlying CaVbeta2 variants, cDNA libraries were constructed from poly(A)(+) RNA isolated from hearts of 7-day-old and adult mice. Screening identified 60 independent CaVbeta2 cDNA clones coding for four types of CaVbeta2 proteins only differing in their 5' sequences. CaVbeta2-N1, -N4, and -N5 but not -N3 were identified in isolated cardiomyocytes by RT-PCR and were sufficient to reconstitute the CaVbeta2 protein pattern in vitro. Significant L-type Ca(2+) currents (I(Ca)) were recorded in HEK293 cells after co-expression of CaV1.2 and CaVbeta2. Current kinetics were determined by the type of CaVbeta2 protein, with the approximately 72-kDa CaVbeta2a-N1 shifting the activation of I(Ca) significantly to depolarizing potentials compared with the other CaVbeta2 variants. Inactivation of I(Ca) was accelerated by CaVbeta2a-N1 and -N4, which also lead to slower activation compared with CaVbeta2a-N3 and -N5. In summary, this study reveals the molecular LTCC composition in mouse heart and indicates that expression of various CaVbeta2 proteins may be used to adapt the properties of LTCCs to changing myocardial requirements during development and that CaVbeta2a-N1-induced changes of I(Ca) kinetics might be essential in embryonic heart.
