Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper

Authors


  • This work was made possible by a number of different awards including two awards supported by the Natural and Environmental Research Council under the ROPA scheme (GR3/R9694) and a PhD studentship GT04/99/MS/301.
  • The nucleotide sequence data reported appear in the EMBL and GenBank nucleotide databases under the accession numbers AJ005834 Fucus vesiculosus MT, KC515393 (full-length fVHA-A) and KC515394 (truncated variant tVHA-A) Fucus vesiculosus V-ATPase A and X98885 Fucus vesiculosus actin gene.

Abstract

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

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