Y3IP1, A nucleus-encoded Thylakoid Protein, Cooperates with the Plastid-Encoded YCf3 Protein in Photosystem I Assembly of Tobacco and Arabidopsis

The intricate assembly of photosystem I (PSI), a large multiprotein complex in the thylakoid membrane, depends on auxiliary protein factors. One of the essential assembly factors for PSI is encoded by ycf3 (hypothetical chloroplast reading frame number 3) in the chloroplast genome of algae and higher plants. To identify novel factors involved in PSI assembly, we constructed an epitope-tagged version of ycf3 from tobacco (Nicotiana tabacum) and introduced it into the tobacco chloroplast genome by genetic transformation. Immunoaffinity purification of Ycf3 complexes from the transplastomic plants identified a novel nucleus-encoded thylakoid protein, Y3IP1 (for Ycf3-interacting protein 1), that specifically interacts with the Ycf3 protein. Subsequent reverse genetics analysis of Y3IP1 function in tobacco and Arabidopsis thaliana revealed that knockdown of Y3IP1 leads to a specific deficiency in PSI but does not result in loss of Ycf3. Our data indicate that Y3IP1 represents a novel factor for PSI biogenesis that cooperates with the plastid genome-encoded Ycf3 in the assembly of stable PSI units in the thylakoid membrane

Toxoplasma ceramide synthases: Gene duplication, functional divergence, and roles in parasite fitness.

Toxoplasma gondii is an obligate, intracellular apicomplexan protozoan parasite of both humans and animals that can cause fetal damage and abortion and severe disease in the immunosuppressed. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localization and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Interestingly, we observed that while TgCerS1 was a fully functional orthologue of the yeast ceramide synthase (Lag1p) capable of catalyzing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow-growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilizing the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this "pseudo" ceramide synthase, TgCerS2, has a considerably greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa may have arisen through gene duplication. However, in the Apicomplexa the duplicated copy is hypothesized to have subsequently evolved into a non-functional "pseudo" ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites. [Abstract copyright: © 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.