Involvement in Fertilization and Expression of Gamete Ubiquitin-Activating Enzymes UBA1 and UBA6 in the Ascidian Halocynthia roretzi.

The extracellular ubiquitin-proteasome system is involved in sperm binding to and/or penetration of the vitelline coat (VC), a proteinaceous egg coat, during fertilization of the ascidian (Urochordata) Halocynthia roretzi. It is also known that the sperm receptor on the VC, HrVC70, is ubiquitinated and degraded by the sperm proteasome during the sperm penetration of the VC and that a 700-kDa ubiquitin-conjugating enzyme complex is released upon sperm activation on the VC, which is designated the "sperm reaction". However, the de novo function of ubiquitin-activating enzyme (UBA/E1) during fertilization is poorly understood. Here, we show that PYR-41, a UBA inhibitor, strongly inhibited the fertilization of H. roretzi. cDNA cloning of UBA1 and UBA6 from H. roretzi gonads was carried out, and their 3D protein structures were predicted to be very similar to those of human UBA1 and UBA6, respectively, based on AlphaFold2. These two genes were transcribed in the ovary and testis and other organs, among which the expression of both was highest in the ovary. Immunocytochemistry showed that these enzymes are localized on the sperm head around a mitochondrial region and the follicle cells surrounding the VC. These results led us to propose that HrUBA1, HrUBA6, or both in the sperm head mitochondrial region and follicle cells may be involved in the ubiquitination of HrVC70, which is responsible for the fertilization of H. roretzi.

Complete Replacement of the Galactolipid Biosynthesis Pathway with a Plant-Type Pathway in the Cyanobacterium Synechococcus elongatus PCC 7942.

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the major components of thylakoid membranes and well-conserved from cyanobacteria to chloroplasts. However, cyanobacteria and chloroplasts synthesize these galactolipids using different pathways and enzymes, but they are believed to share a common ancestor. This fact implies that there was a replacement of the cyanobacterial galactolipid biosynthesis pathway during the evolution of a chloroplast. In this study, we first replaced the cyanobacterial MGDG biosynthesis pathway in a model cyanobacterium, Synechococcus elongatus PCC 7942, with the corresponding plant-type pathway. No obvious phenotype was observed under the optimum growth condition, and the content of membrane lipids was not largely altered in the transformants. We next replaced the cyanobacterial DGDG biosynthesis pathway with the corresponding plant-type pathway using the strain described above and isolated the strain harboring the replaced plant-type pathway instead of the whole galactolipid biosynthesis pathway. This transformant, SeGPT, can grow photoautotrophically, indicating that cyanobacterial galactolipid biosynthesis pathways can be functionally complemented by the corresponding plant-type pathways and that the lipid products MGDG and DGDG, and not biosynthesis pathways, are important. While SeGPT does not show strong growth retardation, the strain has low cellular chlorophyll content but it retained a similar oxygen evolution rate per chlorophyll content compared with the wild type. An increase in total membrane lipid content was observed in SeGPT, which was caused by a significant increase in DGDG content. SeGPT accumulated carotenoids from the xanthophyll groups. These results suggest that cyanobacteria have the capacity to accept other pathways to synthesize essential components of thylakoid membranes.