Isolation And Characterization Of A Functionally Active Protein Translocation Apparatus From Chloroplast Envelopes

Abstract

Chloroplast structure and function depends vitally on the import of nuclear coded and cytosolically synthesized polypeptide constituents (de Boer and Weisbeek 1991). Proteins of the outer and inner envelope from chloroplasts collaborate to form an import machinery which is responsible for the specific recognition of chloroplast destined precursor proteins and their translocation through the two membrane barrier. Outer envelope membrane vesicles are purified from pea chloroplasts in a right side-out orientation, i.e. like in the intact organelle (Waegemann et al. 1992). Precursor proteins are bound to the membrane vesicles in an ATP, receptor and transitpeptide dependent manner (Waegemann and Soll 1991, Soll and Waegemann 1992). The translocation process of a precursor proceeds via distinct steps which can be detected in vitro as translocation intermediates, named deg 1–4 in chloroplasts (Fig 1). The outer envelope localized import apparatus yields deg 1 and 2 while deg 3 and 4 are translocation intermediates which occur in connection with the inner envelope import machinery (Fig 1).