The diatom Phaeodactylum tricornutum harbors a plastid that is surrounded by four membranes and evolved by way of secondary endosymbiosis. Like land plants, most of its plastid proteins are encoded as preproteins on the nuclear genome of the host cell and are resultantly redirected into the organelle. Because two more membranes are present in diatoms than the one pair surrounding primary plastids, the targeting situation is obviously different and more complex. In this work, we focus on preprotein transport across the second outermost plastid membrane -- an issue that was experimentally inaccessible until now. We provide first indications that our hypothesis of an ERAD (ER-associated degradation)-derived preprotein transport system might be correct. Our data demonstrate that the symbiont-specific Der1 proteins, sDer1-1 and sDer1-2, form an oligomeric complex within the second outermost membrane of the complex plastid. Moreover, we present first evidence that the complex interacts with transit peptides of preproteins being transported across this membrane into the periplastidal compartment but not with transit peptides of stromal-targeted proteins. Thus, the sDer1 complex might have an additional role in discriminating preproteins that are transported across the two outermost membranes from preproteins directed across all four membranes of the complex plastid. Altogether, our studies of the symbiont-specific ERAD-like machinery of diatoms suggest that a preexisting cellular machinery was recycled to fulfill a novel function during the transition of a former free-living eukaryote into a secondary endosymbiont.

ABSTRACT. Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus-encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus-encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre-existing ones to new requirements in secondary plastids.

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.

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