| Abstract: | Diatoms and related algae have plastids that are surrounded by four membranes. The outer two membranes are continuous with the endoplasmic reticulum and the inner two membranes are analogous to the plastid envelope membranes of higher plants and green algae. Thus the plastids are completely compartmentalized within the ER membranes. The targeting presequences for nuclear‐encoded plastid proteins have two recognizable domains. The first domain is a classic signal sequence, which presumably targets the proteins to the endoplasmic reticulum. The second domain has characteristics of a transit peptide, which targets proteins to the plastids of higher plants. To characterize these targeting domains, the presequence from the nuclear‐encoded plastid protein AtpC was utilized. A series of deletions of this presequence were fused to Green Fluorescent Protein (GFP) and transformed into cells of the diatom, Phaeodactylum tricornutum. The intracelluar localization of GFP was visualized by fluorescence microscopy. This work demonstrates that the first domain of the presequence is responsible for targeting proteins to the ER lumen and is the essential first step in the plastid protein import process. The second domain is responsible to directing proteins from the ER and through the plastid envelope and only a short portion of the transit peptide‐like domain is necessary to complete this second processing step. In vivo data generated from this study in a fully homologous transformation system has confirmed Gibbs' hypothesis regarding a multistep import process for plastid proteins in chromophytic algae. |
