In Vitro Protein Import by Isolated Chloroplasts

In Chapter 28 , a method for the isolation of intact chloroplasts was described. The availability of this technique has allowed researchers the opportunity to investigate the mechanisms of chloroplast biogenesis. The chloroplast is a complex organelle, being made up of six subcompartments. The thylakoid membrane, which makes up about half the chloroplast, is the site of photosynthetic electron transport, a process involving four multimeric protein complexes (1 ). The majority of chloroplast proteins are nuclear-encoded and synthesized in the cytosol; therefore, there must be some mechanism that allows proteins to cross the chloroplast envelope (the membrane that delineates the organelle) yet also discriminates between the plastid and other organelles, like mitochondria (2 ). Early work in the then embryonic field of chloroplast targeting demonstrated that nuclear-encoded chloroplast proteins were synthesized as precursor proteins. This precursor was processed to the mature form when the precursor was taken up by isolated chloroplasts. This lead to the proposal that the precursor protein interacted with a carrier protein (i.e., a receptor) on the chloroplast envelope that allowed import into the organelle (3 ). Subsequent research demonstrated that an N-terminal extension, termed the transit sequence, present in the precursor, was absolutely required for chloroplast import (reviewed in 2 ; see also Chapter 30 , this volume).