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 in most mammalian cell types. Previous biochemical and immunolocalization studies have revealed the presence of G-protein coupled receptors and heterotrimeric G-protein subunits (Galpha and Gbetagamma subunits) within these structures, implicating mammalian LDTI membrane domains in G-protein coupled signaling. Here, we present biochemical evidence that similar LDTI structures exist in a genetically tractable organism, the yeast Saccharomyces cerevisiae. Yeast LDTI membranes were purified based on the known biochemical properties of mammalian LDTI membranes: (i) their Triton X-100 insolubility; and (ii) their discrete buoyant density in sucrose gradients. As with purified mammalian LDTI membranes, these yeast LDTI membranes harbor the subunits of the heterotrimeric G-proteins (Galpha and Gbetagamma subunits). Other plasma membrane marker proteins (the plasma membrane H+-ATPase and a GPI-linked protein Gas1p) are preferentially excluded from these purified fractions. Mutational and genetic analyses were performed to define the requirements for the targeting of G-protein subunits to these yeast membrane domains. We find that the targeting of Galpha is independent of myristoylation, whereas targeting of Ggamma requires prenylation. Perhaps surprisingly, the targeting of Gbeta to this membrane domain did not require coexpression of Ggamma. It should now be possible to dissect the function of LDTI membrane domains using yeast as a model genetic system.
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The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142-1479, USA. lisanti@wi.mit.edu
Dohlman
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D002451:Cell Compartmentation; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D017830:Octoxynol; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D015398:Signal Transduction; D012995:Solubility
D005656:Fungal Proteins; D017830:Octoxynol; D019204:GTP-Binding Proteins