Nuclear‐encoded Protein Import into Chloroplasts: Methods

Abstract

Chloroplasts carry out essential processes, for example photosynthesis. They are derived from an endosymbiotic event, in which an ancient cyanobacterium was engulfed by an eukaryotic ancestor. During evolution, most of the genes have been transferred to the nucleus. Consequently, >98% of all plastid proteins are translated on cytosolic ribosomes and posttranslationally targeted to and imported into the organelle. Targeting is assisted by cytosolic proteins, which keep them in an import‐competent state. At the chloroplast, many proteins have to conquer the barrier of the chloroplast envelopes. This process is mediated by two complexes in the outer (Toc) and inner (Tic) envelope. Most proteins destined for inner compartments contain a cleavable N ‐terminal transit peptide, whereas most outer envelope components insert into the membrane without such a targeting peptide. Investigation of the import of nuclear‐encoded proteins into chloroplasts is an essential field of plant science. To throw light on this very basic process many different methods have been established. Key Concepts: Studying the in vitro import behaviour of chloroplastic preproteins gives essential insight into this fundamental process. Analysis of mutants in model organisms provides information about the in vivo function of proteins proposed to be involved into translocation.