Abstract
Diatoms are microalgae that possess so-called "complex plastids," which evolved by secondary endosymbiosis and are surrounded by four membranes. Thus, in contrast to primary plastids, which are surrounded by only two membranes, nucleus-encoded proteins of complex plastids face additional barriers, i.e., during evolution, mechanisms had to evolve to transport preproteins across all four membranes. This study reveals that there exist glycoproteins not only in primary but also in complex plastids, making transport issues even more complicated, as most translocation machineries are not believed to be able to transport bulky proteins. We show that plastidal reporter proteins with artificial N-glycosylation sites are indeed glycosylated during transport into the complex plastid of the diatom Phaeodactylum tricornutum. Additionally, we identified five endogenous glycoproteins, which are transported into different compartments of the complex plastid. These proteins get N-glycosylated during transport across the outermost plastid membrane and thereafter are transported across the second, third, and fourth plastid membranes in the case of stromal proteins. The results of this study provide insights into the evolutionary pressure on translocation mechanisms and pose unique questions on the operating mode of well-known transport machineries like the translocons of the outer/inner chloroplast membranes (Toc/Tic).