Title

Identification and molecular characterization of vacuolar type H+-ATPase subunit A and subunit B isoforms in Arabidopsis thaliana

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Botany|Biology, Genetics

Degree

Ph.D.

Abstract

Vacuolar type H+-ATPases are large, multimeric enzyme complexes responsible for the energization of intracellular membranes and the acidification of compartments within eukaryotic cells at the expense of ATP. Subunits A and B are both found in the peripheral membrane sector and are present in three copies per holoenzyme. Subunit A is the catalytic subunit responsible for binding and hydrolyzing ATP. Subunit B is often referred to as the regulatory subunit. The exact function of subunit B in the holoenzyme is not known at this time. ^ Subunit A of the vacuolar type H+-ATPase appears to be present as a single gene in Arabidopsis thaliana. A subunit A cDNA was cloned and sequenced in its entirety. This cDNA translated into a putative protein of 623 amino acids with a molecular weight of 68.7 kDa and a high degree of nucleotide and amino acid identity to subunit genes from other organisms including plants and animals. The characterized subunit gene was shown to utilize at least four different poly (A) tail attachment sites. These different transcripts did not shown any differential expression in response to environmental stress in seedlings. Moreover, the subunit A gene was shown to be expressed in all organs tested and to be upregulated in response to salt and chilling stress. ^ Subunit B of the vacuolar type H+-ATPase exists as a small multigene family of at least three members in Arabidopsis thaliana . Whole genome blot analysis confirmed the existence of three subunit B genes in the Arabidopsis genome. The three genes show a high degree of nucleotide and putative amino acid identity over the small amount of sequence information available as EST's. Subunit B isoforms B-1 and B-2 were shown to be expressed in all organs tested including seedlings, roots, leaves, bolts, siliques, and flowers. Isoform B-3 was expressed in all of the above organs except siliques. The subunit B isoforms showed differential expression in response to environmental stress conditions. Isoforms B-2 and B-3 showed a decrease in transcript levels due to etiolation while B-1 was unchanged. All three showed upregulation due to salt and chilling stress. ^