Kinetic analysis of the transport of thylakoid lumenal proteins in experiments using intact chloroplasts

Abstract

The transport of the lumenal protein plastocyanin has been proposed to occur in two steps: 1) transport across the chloroplastic envelope to the stroma and 2) transport across the thylakoid membrane to the lumen where proteolytic maturation occurs. A partially processed stromal form of plastocyanin has been tentatively identified as a pathway intermediate and as the substrate for the second translocation step (Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. (1986) Cell 46, 365-375). In this study, we have examined the transport kinetics of several lumenal proteins under various incubation conditions. Soluble intermediate sized forms were observed in import reactions with plastocyanin precursors from three higher plant species and with the precursor of the 33-kDa polypeptide of the oxygen-evolving enhancer complex. The accumulation patterns observed for these soluble intermediate sized forms depended on incubation conditions and could not be consistently explained by a simple model where the intermediate sized form is the substrate for the second step. Thus, it has not been possible to clearly identify the substrate for thylakoid translocation in organello.