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Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone.

ABSTRACT: We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots of HvCBT1 showed the presence of six putative transmembrane domains, but sequence alignment indicated a pore domain that was unlike the consensus domains in K+ and olfactory channels. Expression of a subclone of amino acids 482-702 in Escherichia coli generated a peptide that bound CaM. When a fusion protein of HvCBT1 and green fluorescent protein was expressed in barley aleurone protoplasts, fluorescence accumulated in the plasma membrane. Expression of HvCBT1 in the K+ transport deficient Saccharomyces cerevisiae mutant CY162 showed no rescue of the mutant phenotype. However, growth of CY162 expressing HvCBT1 with its pore mutated to GYGD, the consensus sequence of K+ channels, was compromised. We interpret these data as indicating that HvCBT1 acts to interfere with ion transport.

SUBMITTER: Schuurink RC

PROVIDER: S-EPMC19218 | biostudies-literature | 1998 Feb

REPOSITORIES: biostudies-literature

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Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone.

Schuurink R C RC Shartzer S F SF Fath A A Jones R L RL

Proceedings of the National Academy of Sciences of the United States of America 19980201 4

We have used Arabidopsis calmodulin (CaM) covalently coupled to horseradish peroxidase to screen a barley aleurone cDNA expression library for CaM binding proteins. The deduced amino acid sequence of one cDNA obtained by this screen was shown to be a unique protein of 702 amino acids with CaM and cyclic nucleotide binding domains at the carboxyl terminus and high similarity to olfactory and K+ channels. This cDNA was designated HvCBT1 (Hordeum vulgare CaM binding transporter). Hydropathy plots o ...[more]

PMID: 9465122

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Project description:BackgroundAmong natural populations, there are different colours of barley (Hordeum vulgare L.). The colour of barley grains is directly related to the accumulation of different pigments in the aleurone layer, pericarp and lemma. Blue grain colour is due to the accumulation of anthocyanins in the aleurone layer, which is dependent on the presence of five Blx genes that are not sequenced yet (Blx1, Blx3 and Blx4 genes clustering on chromosome 4HL and Blx2 and Blx5 on 7HL). Due to the health benefits of anthocyanins, blue-grained barley can be considered as a source of dietary food. The goal of the current study was to identify and characterize components of the anthocyanin synthesis regulatory network for the aleurone layer in barley.ResultsThe candidate genes for components of the regulatory complex MBW (consisting of transcription factors MYB, bHLH/MYC and WD40) for anthocyanin synthesis in barley aleurone were identified. These genes were designated HvMyc2 (4HL), HvMpc2 (4HL), and HvWD40 (6HL). HvMyc2 was expressed in aleurone cells only. A loss-of-function (frame shift) mutation in HvMyc2 of non-coloured compared to blue-grained barley was revealed. Unlike aleurone-specific HvMyc2, the HvMpc2 gene was expressed in different tissues; however, its activity was not detected in non-coloured aleurone in contrast to a coloured aleurone, and allele-specific mutations in its promoter region were found. The single-copy gene HvWD40, which encodes the required component of the regulatory MBW complex, was expressed constantly in coloured and non-coloured tissues and had no allelic differences. HvMyc2 and HvMpc2 were genetically mapped using allele-specific developed CAPS markers developed. HvMyc2 was mapped in position between SSR loci XGBS0875-4H (3.4 cM distal) and XGBM1048-4H (3.4 cM proximal) matching the region chromosome 4HL where the Blx-cluster was found. In this position, one of the anthocyanin biosynthesis structural genes (HvF3'5'H) was also mapped using an allele-specific CAPS-marker developed in the current study.ConclusionsThe genes involved in anthocyanin synthesis in the barley aleurone layer were identified and characterized, including components of the regulatory complex MBW, from which the MYC-encoding gene (HvMyc2) appeared to be the main factor underlying variation of barley by aleurone colour.

Project description:As a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a unique Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. TRPV5-mediated Ca2+ entry into the cell is controlled by a negative feedback mechanism, in which calmodulin (CaM) blocks the TRPV5 pore upon Ca2+ binding. Combining microscopy techniques and biochemical assays, the present study uncovered an auxiliary role for CaM in the regulation of human (h)TRPV5 intracellular trafficking. Overexpressed hTRPV5 was mainly localised to the endoplasmic reticulum (ER) and associated with peripheral ER tubules. Limiting expression using the HEK293 TET-off system revealed that hTRPV5 trafficked through the endocytic recycling pathway. CaM co-localised with hTRPV5 at intracellular sites and overexpression of CaM slowed hTRPV5 exit from the ER. In accordance, CaM binding-disrupting truncations of the TRPV5 C-terminus (698X) or knockdown of endogenous CaM by small interfering RNA resulted in an increased fraction of TRPV5 that localised to the plasma membrane. hTRPV5 expressing cells had an increased intracellular Ca2+ concentration upon knockdown of CaM. The protein abundance of the Ca2+ impermeable hTRPV5-D542 mutant is also regulated by CaM, which suggests that the mode of action is independent of disrupted intracellular calcium concentrations. In conclusion, our study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, modulating TRPV5 intracellular trafficking. KEY POINTS: The renal Ca2+ channel TRPV5 is a crucial player in maintenance of the body's Ca2+ homeostasis. Ca2+ transport through TRPV5 is controlled by single channel activity, as well as TRPV5 plasma membrane abundance. Calmodulin (CaM) co-localised with TRPV5 at intracellular sites and retained TRPV5 in the endoplasmic reticulum. Disrupted CaM-TRPV5 binding or knockdown of endogenous CaM by small interfering RNA (siRNA) resulted in an increased TRPV5 plasma membrane abundance. Knockdown of endogenous CaM by siRNA resulted in increased intracellular Ca2+ concentrations. The regulation of TRPV5 trafficking by CaM is independent of the effect of CaM on intracellular Ca2+ concentrations. This study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, next to its ability to directly block the TRPV5 channel pore, by modulating TRPV5 trafficking in the secretory pathway.

Dataset Information

Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone.

Publications

Characterization of a calmodulin-binding transporter from the plasma membrane of barley aleurone.

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