Topological organization of the hyaluronan synthase from Streptococcus pyogenes

Since we first reported (DeAngelis, P, L,, Papaconstantinou, J,, and Weigel , P, H. (1993) J, Biol. Chem. 268, 19181-19184) the cloning of the hyaluron an (HA) synthase from Streptococcus pyogenes (spHAS), numerous membrane-bou nd HA synthases have been discovered in both prokaryotes and eukaryotes. Th e HASs are unique among enzymes studied to date because they mediate 6-7 di screte functions in order to assemble a polysaccharide containing hetero-di saccharide units and simultaneously effect translocation of the growing HA chain through the plasma membrane. To understand how the relatively small s pHAS performs these various functions, we investigated the topological orga nization of the protein utilizing fusion analysis with two reporter enzymes , alkaline phosphatase and beta -galactosidase, as well as several other ap proaches. From these studies, we conclude that the NH2 terminus and the COO H terminus, as well as the major portion of a large central domain are loca lized intracellularly, The first two predicted membrane domains were confir med to be transmembrane domains and give rise to a very small extracellular loop that is inaccessible to proteases, Several regions of the large inter nal central domain appear to be associated with, but do not traverse, the m embrane. Following the central domain, there are two additional transmembra ne domains connected by a second small extracellular loop that also is inac cessible to proteases, The COOH-terminal similar to 25% of spHAS also conta ins a membrane domain that does not traverse the membrane and may contain e xtensive re-entrant loops or amphipathic helices, Numerous membrane associa tions of this latter COOH-terminal region and the central domain may be req uired to create a pore-like structure through which a growing HA chain can be extruded to the cell exterior. Based on the high degree of similarity am ong Class I HAS family members, these enzymes may have a similar topologica l organization for their spHAS-related domains.