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11 Páginas.-- 5 Figuras, Carotenoids are isoprenoid lipids found across the tree of life with important implications in oxidative stress adaptations, photosynthetic metabolisms, as well as in membrane dynamics. The canonical view is that C40 carotenoids are synthesized from phytoene and C30 carotenoids from diapophytoene. Squalene is mostly associated with the biosynthesis of polycyclic triterpenes, although there have been suggestions that it could also be involved in the biosynthesis of C30 carotenoids. However, demonstration of the existence of this pathway in nature is lacking. Here, we demonstrate that C30 carotenoids are synthesized from squalene in the Planctomycetes bacteria and that this squalene route to C30 carotenoids is the most widespread in prokaryotes. Using the evolutionary history of carotenoid and squalene amino oxidases, we propose an evolutionary scenario to explain the origin and diversification of the different carotenoid and squalene-related pathways. We show that carotenoid biosynthetic pathways have been constantly transferred and neofunctionalized during prokaryotic evolution. One possible origin of the squalene pathway connects it with the one of C40 carotenoid synthesis of Cyanobacteria. The widespread occurrence of the squalene route to C30 carotenoids in Bacteria increases the functional repertoire of squalene, establishing it as a general hub of carotenoids and polycyclic triterpenes synthesis., This work was supported by the Spanish Ministry of Economy and Competitiveness (Grant No. BFU2016-78326-Pand and MDM-2016-0687). This research is funded in part by the Gordon and Betty Moore foundation through Grant GBMF9733 to D.P.D. We also thank Berend Snel and Anja Spang for financial support of C.S.-M. through a grant funded by Utrecht University and the Netherlands Organisation for Scientific Research (NWO).

The nuclear pore complex (NPC) is the sole mediator of transport between the nucleus and the cytoplasm. The NPC is composed of about 30 distinct proteins, termed nucleoporins or nups. The yeast (Rout et al., J Cell Biol 148:635-651, 2000) and mammalian (Cronshaw et al., J Cell Biol 158:915-927, 2002) NPC have been extensively studied. However, the two species are relatively closely related. Thus, to reveal details about NPC evolution, we chose to characterize the NPC of a distantly related organism, Trypanosoma brucei. We took a subcellular proteomic approach and used several complementary strategies to identify 865 proteins associated with the nuclear envelope. Over 50% of ∼ 8,100 open reading frames of T. brucei have little or no known function because T. brucei is distantly related to model metazoa and fungi (Berriman et al., Science 309:416-422, 2005). By sequence similarity alone, we could identify only five nucleoporins. This chapter outlines our strategy to identify 17 additional nucleoporins as well as contribute functional annotation data to the T. brucei genome database.

Multiprotein complexes are key players in virtually all important cellular processes. The past year has seen the publication of several papers that have illuminated what we know about the number and composition of these molecular machines, using high-throughput purification methods. Other studies have illuminated structural and functional aspects of protein interactions, networks and molecular assemblies. As a result, we have a more complete view of how many complexes are in living systems, what they look like and the roles they play in the cell.

We examined the microenvironment of the single tryptophan and the tyrosine residues of PsbQ, one of the three main extrinsic proteins of green algal and higher plant photosystem II. On the basis of this information and the previous data on secondary structure [Balsera, M., Arellano, J.B., Gutiérrez, J.R., Heredia, P., Revuelta, J.L.De Las Rivas, J. (2003) Biochemistry42, 1000-1007], we screened structural models derived by combining various threading approaches. Experimental results showed that the tryptophan residue is partially buried in the core of the protein but still in a polar environment, according to the intrinsic fluorescence emission of PsbQ and the fact that fluorescence quenching by iodide was weaker than that by acrylamide. Furthermore, quenching by cesium suggested that a positively charged barrier shields the tryptophan microenvironment. Comparison of the absorption spectra in native and denaturing conditions indicated that one or two out of six tyrosines of PsbQ are buried in the core of the structure. Using threading methods, a 3D structural model was built for the C-terminal domain of the PsbQ protein family (residues 46-149), while the N-terminal domain is predicted to have a flexible structure. The model for the C-terminal domain is based on the 3D structure of cytochrome b562, a mainly alpha-protein with a helical up/down bundle folding. Despite the large sequence differences between the template and PsbQ, the structural and energetic parameters for the explicit model are acceptable, as judged by the corresponding tools. This 3D model is compatible with the experimentally determined environment of the tryptophan residue and with published structural information. The future experimental determination of the 3D structure of the protein will offer a good validation point for our model and the technology used. Until then, the model can provide a starting point for further studies on the function of PsbQ.