Experiments on fatty acids chain elongation and glycan flipping in the ER membrane

Abstract Very long chain fatty acids (VLCFA) are essential molecules that take part in many different cellular processes such as membrane pore stabilization, membrane trafficking and signaling pathways. The fatty acid elongation pathway in yeast has been studied for about a decade. As part of our work on cellular VLCFA elongation, we identified and characterized the condensing enzyme as well as ketoacyl reductases of the elongation pathway in cotton. In order to identify the yeast 3-hydroxyacyl-CoA dehydratase, we introduced a redundancy in this function by engineering a chimera consisting of the two first predicted transmembrane domains of Elo3p and the hydratase2 domain of Candida tropicalis Mfe2p. Yeast harboring the chimeric construct were subjected to random mutagenesis, and screened for mutants whose survival was dependent on the chimera. The mutants isolated contained RFT1 mutations and exhibited a defect in protein glycosylation, but no VLCFA deficiencies. The N-linked glycosylation pathway is well conserved in eukaryotes. Glycan synthesis occurs on the ER membrane; first on the cytoplasmic side up to Dol-PP-GlcNAc2Man5, which is then translocated to the ER luminal side in an Rft1p-dependent flipping process. The core glycan is further extended to Dol-PP-GlcNAc2Glc3Man9, and then transferred to an asparagine side chain of the nascent polypeptide to be glycosylated. It was found that the Elo3'-hydratase2 chimera acts as a multicopy suppressor of the Rft1p deficiency. The subsequent studies elucidated new aspects of Rft1p function, as well as a hitherto under-appreciated role of the ER associated protein degradation process in the maintenance of ER integral membrane complexes and the physical integrity of the membrane. The functionality of the human Rft1p homologue was demonstrated using a yeast complementation assay. A mutant variant from a patient was analyzed, aiding in the identification and characterization of the first reported case of a glycosylation deficiency in humans caused by a defective RFT1 allele