Identification and subcellular localization of the soybean copper P1B-ATPase GmHMA8 transporter

Abstract

We have identified a copper P(1B)-ATPase transporter in soybean (Glycine max), named as GmHMA8, homologue to cyanobacterial PacS and Arabidopsis thaliana AtHMA8 (PAA2) transporters. A novel specific polyclonal anti-GmHMA8 antibody raised against a synthetic peptide reacted with a protein of an apparent mass of around 180-200 kDa in chloroplast and thylakoid membrane preparations isolated from soybean cell suspensions. Immunoblot analysis with this antibody also showed a band with similar apparent molecular mass in chloroplasts from Lotus corniculatus. Immunofluorescence labelling with the anti-GmHMA8 antibody and double immunofluorescence labelling with anti-GmHMA8 and anti-RuBisCo antibodies revealed the localization of the GmHMA8 transporter within the chloroplast organelle. Furthermore, the precise ultrastructural distribution of GmHMA8 within the chloroplast subcompartments was demonstrated by using electron microscopy immunogold labelling. The GmHMA8 copper transporter from soybean was localized in the thylakoid membranes showing a heterogeneous distribution in small clusters.