9.B.119 The Glycan Synthase, Fks1 (Fks1) Family
Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hotspots 1, 2 and 3 (residues 635-649, 1354-1361, and 690-700, respectively). Johnson and Edlind (2012) used HA-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology, and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 TMSs predicted for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N-terminus (residues 1-445) is cytosolic and the C-terminus (residues 1823-1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715-1294) includes homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hotspots are external, with hotspot 1 adjacent to and hotspot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hotspot 3 and through their cyclic peptides with hotspots 1 and 2 (Johnson and Edlind, 2012). A transport function is possible but not identified.