Identification of a molybdenum uptake pump in Arabidopsis plants

Heavy metal pumps belong to the P-type ATPase family. They have been found in all phylogenetic kingdoms, and have been implicated in heavy metal resistance and regulation of essential micronutrients.; Here I report the cloning and characterization of a heavy metal pump from Arabidopsis, called AMA1 (A&barbelow;rabidopsis Heavy M&barbelow;etal A&barbelow;TPase-isoform 1&barbelow;). AMA1 encodes a 102 kDa P-type ATPase (AMA1p). Based on hydropathy analysis, eight transmembrane domains are predicted, with a "CPC" motif, characteristic of heavy metal P-type ATPases, located in the putative transmembrane domain 6. AMA1p differs from other heavy metal pumps in that it contains a unique 26kDa C-terminal domain that are rich in histidine and cysteine, with the cysteines commonly found as CC pairs.; AMA1p is localized to the plasma membrane, as indicated by aqueous two-phase partitioning and immunodetection, and by confocal microscopy of plants expressing an AMA1p-GFP fusion protein. Western blot analysis of membrane proteins from different tissues indicates that AMA1p is expressed at a high level in siliques and a medium level in roots and stems.; To genetically investigate the biological function of AMA1p, I identified a plant line containing a T-DNA disrupted allele, ama1-1, by a PCR-based screening approach. ama1-1 homozygous plants (ama1-1/ama1-1) grow normally under standard growth conditions. However, metal analysis using Ion Conductive Plasma-Optical Emission Spectrometry (ICP-OES) indicated that ama1-1 plants have 2 to 5-fold less molybdenum than wild-type plants, while other metals are present at normal levels. This reduced molybdenum accumulation is reversed by expressing fusion proteins of AMA1p or AMA1p-DeltaNC (N- and C-terminal truncated AMA1p) fused with GFP.; The results presented here suggest that AMA1p functions as a molybdenum uptake pump in plants. This research provides the first evidence for a P-type ATPase involved in molybdenum transport.; In addition, another P-type ATPase, ACA3 (Arabidopsis Ca2+-ATPase isoform 3), was cloned and characterized. ACA3p has a predicted size of 116 kDa and is most similar to endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+-ATPases in animal cells. Western blot analysis indicates that ACA3p is most abundant in roots and flowers. It is localized to an endomembrane, as indicated by aqueous two-phase partitioning and immunodetection.