Protein Targeting in ‘‘Secondary’’ or ‘‘Complex’’ Chloroplasts

All the algae with chlorophyll (Chl) c (haptophytes, cryptophytes, and heterokonts such as diatoms) acquired their chloroplasts by secondary endosymbiosis, where a nonphotosynthetic eukaryote host engulfed (or was invaded by) a red alga. This resulted in chloroplasts with four bounding membranes. The outermost membrane (chloroplast endoplasmic reticulum [ER]), is physically continuous with the rough ER, and in some algal species can be seen to have cytoplasmic ribosomes attached to its outer surface. All nuclear-encoded chloroplast proteins have an N-terminal ER targeting sequence, which is cleaved off during transit across this membrane. We know little about how proteins cross the next membrane and engage the import translocons of the envelope membranes. One way to study the targeting of proteins across the inner membranes is to make constructs lacking the ER signal sequence, translate them in vitro, and assay their import into pea chloroplasts.