부산대학교 도서관

R3159 --> P1B‐type ATPases are primary active transmembrane metal pumps conserved in all kingdoms of life that play a pivotal role in controlling cellular metal ion levels and homeostasis. In bacteria, they are responsible for selectively exporting essential and toxic metal ions across the cell membrane to maintain concentrations below toxic levels. They are critical virulence factors in pathogenic bacteria and allow selected microorganisms to survive in extreme metal‐rich environments. The P1B‐2‐type Zn(II) ATPase pump subfamily possesses a wide promiscuity towards metal substrate translocation by plastic selection of Zn(II)/Cd(II)/Hg(II)/Pb(II) at their high‐affinity transmembrane metal binding site. Despite these substrates stimulate ATP‐hydrolysis, direct translocation of all these metals and their mechanism of transport remain to be demonstrated. In this work, we established a platform for purification and in‐vitro functional reconstitution in artificial phospholipid bilayer vesicles (proteoliposomes) of ZntA from Cupriavidus metallidurans to dissect the molecular mechanism of divalent metal ion translocation and substrate promiscuity towards first‐, second‐ and the third‐row transition metals. Metal‐dependent ATPase activity assays of CmΔZntA166–794 in detergent micelles and proteoliposomes revealed stimulation of the ATPase activity by Pb(II), Zn(II), Cd(II) and Hg(II) substrates with the maximal velocities (Vmax) following the order Pb(II) > Zn(II) > Cd(II) > Hg(II), confirming the successful functional reconstitution in lipid vesicles. By selecting and encapsulating divalent metal sensitive fluorescent probes in the proteoliposome lumen, we performed real‐time metal transport assays to reveal the ATP‐dependent translocation of Pb(II), Zn(II), Cd(II) and Hg(II) across the lipid bilayer, establishing that the translocation rates correlate to the order observed following ATP hydrolysis. Furthermore, to dissect the substrate transport mechanism, we encapsulated the pH sensitive fluorescent probe pyranine to demonstrated that, differently than other P‐type ATPases, translocation is not coupled to H+ counter‐flux for all the M(II) substrates. By encapsulating the membrane potential sensitive fluorescent probe Oxonol‐VI we also revealed that, with all substrates, Zn‐pumps act as primary active electrogenic uniporters capable of generating a transmembrane potential. This work establishes a platform to dissect the mechanisms of translocation of various metal substrates in P‐type ATPases which is applicable to study other uncharacterized transporter families and to reveal the bioinorganic chemistry of metal ion transport at molecular level. [ABSTRACT FROM AUTHOR]