Inventory of the superfamily of P-type ion pumps in Arabidopsis

Abstract

A total of 45 genes encoding for P-type ATPases have been identified in the complete genome sequence of Arabidopsis. Thus, this plant harbors a primary transport capability not seen in any other eukaryotic organism sequenced so far. The sequences group in all five subfamilies of P-type ATPases. The most prominent subfamilies are P(1B) ATPases (heavy metal pumps; seven members), P(2A) and P(2B) ATPases (Ca(2+) pumps; 14 in total), P(3A) ATPases (plasma membrane H(+) pumps; 12 members including a truncated pump, which might represent a pseudogene or an ATPase-like protein with an alternative function), and P(4) ATPases (12 members). P(4) ATPases have been implicated in aminophosholipid flipping but it is not known whether this is a direct or an indirect effect of pump activity. Despite this apparent plethora of pumps, Arabidopsis appears to be lacking Na(+) pumps and secretory pathway (PMR1-like) Ca(2+)-ATPases. A cluster of Arabidopsis heavy metal pumps resembles bacterial Zn(2+)/Co(2+)/Cd(2+)/Pb(2+) transporters. Two members of the cluster have extended C termini containing putative heavy metal binding motifs. The complete inventory of P-type ATPases in Arabidopsis is an important starting point for reverse genetic and physiological approaches aiming at elucidating the biological significance of these pumps.

Figure 1

Overview of the Arabidopsis P-type ATPase superfamily. Families are designated by numerals on the left followed by gene names. The putative transported ions are indicated on the right. Boxes indicate transmembrane segments; black circles, regulatory domains containing autoinhibitory sequences; white circles, HMA domains; black boxes: CC dipeptide domains; white boxes, poly-His domains. HMA, CC dipeptide, and poly-His domains are putatively involved in heavy metal binding and sensing. Abbreviations are: 14-3-3, 14-3-3 protein binding region; CaM, calmodulin binding region; and PL, phospholipids.

Figure 2

Phylogenetic tree of Arabidopsis P-type ATPases. Conserved segments present in all P-type ATPases were extracted from the sequences and were aligned using T-COFFEE (Notredame et al., 2000). The alignment was used to perform a phylogenetic analysis using the Protdist and Fitch program from the Phylip package (Felsenstein, 1989). The resulting phylogenetic tree reveals five major branches, which are named according to Axelsen and Palmgren (1998).

Figure 3

Alignment of Ca²⁺ binding residues in TM segments from different P-type ATPases. An alignment of the full length Arabidopsis ATPases was performed using T-COFFEE (Notredame et al., 2000). The TM segments 4–6 and 8 were extracted from the alignment. The overall alignment was reliable for TM segments 4–6, whereas the alignment of TM 8 is more dubious as the similarity between the P_2A, P₄ and P₅ ATPases is low in this region. The alignment includes the P_2A ATPase SERCA1a from rabbit (ATC1) the Ca²⁺ ATPases PMR1 (P_2A) and PCA1 (P_2B) from S. cerevisiae, the P_2D Ca²⁺ ATPase CTA3 from Schizosaccharomyces pombe as well as a selection of ATPases from Arabidopsis. Residues involved in coordination of Ca²⁺ in the two Ca²⁺ binding sites (Site I and Site II) found in the 2.6 Å crystal structure of SERCA1a (Toyoshima et al., 2000) are marked with 1 and 2, respectively. The position marked X is a residue involved in coordination of both Ca²⁺ ions. Residues with hydrophobic side chains in TM 4 contribute with backbone carbonyl oxygens.