Trypsination of inside-out chloroplast thylakoid vesicles for localization of the water-splitting site

Abstract

Many conflicting results have been reported concerning the location of the water splitting site in the chloroplast thylakoid membrane [ 1,2]. An internal location has been assumed mainly from the internal release of protons linked to oxygen production [3,4] and the selective release of manganese into the lumenal space upon Tris treatment [S]. In contrast, an external exposure has been inferred from the inactivation of photosystem II electron transport by chemical probes considered to be unable to penetrate the membrane [6,7]. A location at the outer surface has also been concluded from the trypsin digestion of the chloroplast lamellae [8,9] while the effect of trypsin has been claimed to be due mainly to proteolysis of the reducing side of photosystem II [lO,l I]. These conflicting results may reflect the inherent limitations in modifying only the outer membrane surface for studies on the transverse organization of a membrane. Modification of a component on the outer surface may cause rearrangements in the membrane which leads to inactivation of internal catalytic sites. However, the absence of effects does not reveal whether a component is located internally or just shielded behind some bulky membrane constituents. A direct comparison between the effects of non-penetrating reagents on the outer and inner membrane surfaces is therefore desirable. Such