Regulatory mechanisms of plant P-type ATPases

Abstract

The Zea mays plasma membrane H⁺-ATPase gene family consists of at least 11 isoforms as evidenced by the cloning of 11 unique H⁺-ATPase gene fragments. Transcripts for three of these isoforms (MHA2, MHA3, and MHA4) were shown to be expressed in coleoptile segments and root tips by RT-PCR experiments. Curiously, MHA4 transcripts are represented by 2 size classes, and sequence analysis revealed that they are likely derived from alternative splicing. The smaller transcript, MHA4d, is produced by splicing at a non-consensus splice site and is extremely rare. Although a putative MHA4d polypeptide is present in coleoptile and root tip microsomes, definitive evidence that this polypeptide is indeed MHA4d is lacking.

The Arabidopsis endoplasmic reticulum Ca²⁺-ATPase, ACA2, has been shown to be regulated by calmodulin via an N-terminal autoinhibitor (Harper et al., 1998). I have utilized complementation of the yeast strain K616, which cannot grow on Ca²⁺-depleted media, as a genetic tool to identify residues important for autoinhibition in ACA2. Ethyl methane sulfonate (EMS), PCR and site-directed mutagenesis were used to generate random mutations in ACA2 which complement K616. Ten unique point mutations in ACA2 were identified. Seven of these mutations were located in the N-terminal autoinhibitory region and the remaining 3 were located on the cytoplasmic surface of transmembrane domain 1 (M1), within transmembrane domain 2 (M2), and near the stalk region, S3 (E167/K, D219/N, and E341/K, respectively). The latter three mutations have not been previously shown to be important for autoinhibition and were further investigated for selected biochemical parameters. ATPase assays of these mutant enzymes indicated that they were indeed constitutively active (i.e., deregulated). The Km_ATP and Km_Ca2+ of these mutants were similar to those of the N-terminally truncated enzyme lacking the autoinhibitor with the exception of D219/N, which had a 2-fold higher affinity for ATP. Mutations D219/A, D219/K and the conservative mutation D219/E also resulted in deregulated pumps as shown by complementation of K616 and ATPase assays. Collectively, these results suggest that D219 plays an important role in autoinhibition.