IDENTIFICATION OF TRITON X-100 INSOLUBLE MEMBRANE DOMAINS IN THE YEAST SACCHAROMYCES-CEREVISIAE - LIPID REQUIREMENTS FOR TARGETING OF HETEROTRIMERIC G-PROTEIN SUBUNITS

Low density Triton X-100 insoluble (LDTI) membrane domains are found i n most mammalian cell types. Previous biochemical and immunolocalizati on studies have revealed the presence of G-protein coupled receptors a nd heterotrimeric G-protein subunits (G(alpha) and G(beta gamma) subun its) within these structures, implicating mammalian LDTI membrane doma ins in G-protein coupled signaling. Here, we present biochemical evide nce that similar LDTI structures exist in a genetically tractable orga nism, the yeast Saccharomyces cerevisiae. Yeast LDTI membranes were pu rified based on the known biochemical properties of mammalian LDTI mem branes: (i) their Triton X-100 insolubility; and (ii) their discrete b uoyant density in sucrose gradients. As with purified mammalian LDTI m embranes, these yeast LDTI membranes harbor the subunits of the hetero trimeric G-proteins (G(alpha) and G(beta gamma) subunits). Other plasm a membrane marker proteins (the plasma membrane H+-ATPase and a GPI-li nked protein Gas1p) are preferentially excluded from these purified fr actions. Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast me mbrane domains, We find that the targeting of G(alpha) is independent of myristoylation, whereas targeting of G(gamma) requires prenylation. Perhaps surprisingly, the targeting of G(beta) to this membrane domai n did not require coexpression of G(gamma). It should now be possible to dissect the function of LDTI membrane domains using yeast as a mode l genetic system.