Re-examination of the localization of Mg-chelatase within the chloroplast

In a plastid-free assay, Mg-chelatase from pea ( Pisum sativum L. cv. Spring) and cucumber ( Cucumis sativus L. cv. Sumter) chloroplasts is inhibited to equal extents by the mercurial reagents. p -chloromercuribenzoate (PCMB) and p -chloromercuribenzene sulfonate (PCMBS). However, in intact chloroplasts PCMB inhibits Mg-chelatase fourfold more strongly than does PCMBS. Since PCMBS cannot penetrate membranes as readily as PCMB, Mg-chelatase may be localized interior to the inner chloroplast envelope. When Mg-chelatase is assayed with photosynthetically generated ATP, the presence of an external ATP trap does not inhibit activity, suggesting that the enzyme is not located in the interenvelope space. None of the components of Mg-chelatase are integral membrane proteins: Mg-chelatase activity is readily solubilized by washing the total chloroplast membranes in buffers of low MgCl₂ content. This precludes localization by purifying individual thylakoid and envelope membranes which requires low MgCl₂ concentrations.