Endomembrane structure and the chloroplast protein targeting pathway in Heterosigma akashiwo (Raphidophyceae, Chromista)

Chloroplasts in heterokont algae are surrounded by four membranes and proba bly originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host, Understanding how nuclear-encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these memb ranes could give us some insights about how the endosymbiont: was integrate d into the host cell in the process of secondary symbiogenesis. In multipla stid heterokont algae such as raphidophytes, it has been unclear if the out ermost of the four membranes surrounding the chloroplast (the chloroplast e ndoplasmic reticulum [CER] membrane) is continuous with the nuclear envelop e and rough endoplasmic reticulum (ER). Here, we report detailed ultrastruc tural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada exY . Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with t hree envelope membranes is located within the ER lumen, that is, topologica lly the same structure as that of monoplastid heterokont algae. However the CER membrane of II. akashiwo had very few, if any, ribosomes attached, unl ike the CER membranes in other heterokont algae. To verify that proteins ar e first targeted to the ER, we assayed protein import into canine microsome s using a precursor for a nuclear-encoded chloroplast protein, the fucoxant hin-chlorophyll a/c protein of H, akashiwo. This demonstrated that the prec ursor has a functional signal sequence for ER targeting and is cotranslatio nally translocated into the ER, where a signal sequence of about 17 amino a cids is removed, Based on these data, we hypothesize that II H. akashiwo, n uclear-encoded chloroplast protein precursors that have been cotranslationa lly transported into the ER lumen are sorted in the ER and transported to t he chloroplasts through the ER lumen.