ERAD components in organisms with complex red plastids suggest recruitment of a preexisting protein transport pathway for the periplastid membrane

Abstract

The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis.

FIG. 1.—

Plastid architecture of stramenopiles and haptophytes. The secondary plastids of, for example, Phaeodactylum tricornutum and Emiliania huxleyi both have the same membrane organization. They are surrounded by four membranes with the outermost one being contiguous to the ER. Plastid proteins presumably cross the 1st, 2nd, 3rd, and 4th membrane by Sec61, SELMA (investigated in this study), TOC, and TIC translocons, respectively. IMS, intermembrane space.

FIG. 2.—

Import properties of heterologously expressed haptophytic sequences. The topogenic signals of sDer1-1 and sUba1 direct GFP to the PPC. The GFP fusion comprising the targeting signal of sCdc48 predominantly localizes to the ER. The presequences of BiP and AtpG lead to either ER or stromal localization of GFP, respectively. The host versions of Uba1 and Cdc48 localize within the cytosol. Columns from left to right: light microscopic images, chlorophyll autofluorescence, GFP fluorescence and YFP fluorescence, respectively, merged chlorophyll and GFP/YFP fluorescence. Scale bar represents 10 μm.

FIG. 3.—

Phylogenetic bootstrap trees and Neighbor Net (NNet) splits graphs. (a) PHyML bootstrap tree derived from an FSA alignment of Cdc48 sequences from 39 taxa (646 of 2,812 sites were used after curation with Gblocks). Bootstrap analyses were conducted using 100 bootstrap replicates. (b) NNet splits graph derived from an FSA alignment of Cdc48 sequences from 39 taxa (646 of 2812 sites that were left after curation with Gblocks were used for the estimation of distances using PROTDIST and the JTT model). With these distances, a NNet splits graph was constructed which was visualized with Splitstree4. Three splits are highlighted: Split 1 separates the symbiontic sequences from all other taxa. This split also occurs in 82 of 100 constructed bootstrap trees. Split 2 separates the nucleomorph-encoded sequences and the red algal copy from all other taxa, whereas split 3 unites the Emiliania host-derived Cdc48 sequence with its homologues from the cryptophyte nucleus and green plants with a bootstrap support of 68%. (c) PHyML bootstrap tree derived from an FSA alignment of Uba1 sequences from 38 taxa (438 of 7,999 sites were used after curation with Gblocks). Bootstrap analyses were conducted using 100 bootstrap replicates. (d) NNet splits graph derived from an FSA alignment of Uba1 sequences from 38 taxa (438 of 7,999 sites that were left after curation with Gblocks were used for the estimation of distances using PROTDIST and the JTT model). With these distances, a NNet splits graph was constructed which was visualized with Splitstree4. Split 4 separates the green lineage together with the Emiliania huxleyi and the Guillardia theta sequences from all other taxa and has a bootstrap support of 98% in the corresponding ML tree.

FIG. 3.—

Phylogenetic bootstrap trees and Neighbor Net (NNet) splits graphs. (a) PHyML bootstrap tree derived from an FSA alignment of Cdc48 sequences from 39 taxa (646 of 2,812 sites were used after curation with Gblocks). Bootstrap analyses were conducted using 100 bootstrap replicates. (b) NNet splits graph derived from an FSA alignment of Cdc48 sequences from 39 taxa (646 of 2812 sites that were left after curation with Gblocks were used for the estimation of distances using PROTDIST and the JTT model). With these distances, a NNet splits graph was constructed which was visualized with Splitstree4. Three splits are highlighted: Split 1 separates the symbiontic sequences from all other taxa. This split also occurs in 82 of 100 constructed bootstrap trees. Split 2 separates the nucleomorph-encoded sequences and the red algal copy from all other taxa, whereas split 3 unites the Emiliania host-derived Cdc48 sequence with its homologues from the cryptophyte nucleus and green plants with a bootstrap support of 68%. (c) PHyML bootstrap tree derived from an FSA alignment of Uba1 sequences from 38 taxa (438 of 7,999 sites were used after curation with Gblocks). Bootstrap analyses were conducted using 100 bootstrap replicates. (d) NNet splits graph derived from an FSA alignment of Uba1 sequences from 38 taxa (438 of 7,999 sites that were left after curation with Gblocks were used for the estimation of distances using PROTDIST and the JTT model). With these distances, a NNet splits graph was constructed which was visualized with Splitstree4. Split 4 separates the green lineage together with the Emiliania huxleyi and the Guillardia theta sequences from all other taxa and has a bootstrap support of 98% in the corresponding ML tree.