title: Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.
abstract: In complex with the prolyl isomerase FKBP12, the natural product rapamycin blocks signal transduction in organisms as diverse as yeast and man. The yeast targets of FKBP12-rapamycin, TOR1 and TOR2, are large proteins with homology to lipid and protein kinases. A mammalian FKBP12-rapamycin binding protein, RAFT1, shares 39% and 43% identity with TOR1 and TOR2 proteins, respectively but has not been linked to rapamycin action in vivo. We find that when expressed in yeast, neither wild-type nor mutant RAFT1 complemented tor mutations or conferred rapamycin resistance. In contrast, TOR1-RAFT1 and TOR1-RAFT1 hybrid proteins containing the carboxy-terminal RAFT1 kinase domain complemented tor2 and tor1 mutant strains, respectively. Moreover, TOR2-RAFT1 and TOR1-RAFT1 hybrid proteins mutated at the position corresponding to rapamycin-resistant TOR mutants (S20351) conferred rapamycin resistance. Like the TOR2 protein, the TOR2-RAFT1 proteins were stably expressed, localized to the vacuolar surface, and associated with a phosphatidylinositol-4 kinase activity. These findings directly link the mammalian TOR homolog RAFT1 to rapamycin action in vivo and indicate that the TOR/RAFT1 kinase domain has been functionally conserved from yeast to man.
authors.affiliation: Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710 USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Alarcon
authors.forename: M E
authors.initials: ME
authors.lastname: Cardenas
authors.forename: J
authors.initials: J
authors.lastname: Heitman
mesh_terms: D019870:1-Phosphatidylinositol 4-Kinase; D000818:Animals; D000935:Antifungal Agents / Q000494:pharmacology; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D018797:Cell Cycle Proteins; D002462:Cell Membrane / Q000737:chemistry; D004352:Drug Resistance, Microbial; D005656:Fungal Proteins / Q000502:physiology*; D005816:Genetic Complementation Test; D008322:Mammals; D008969:Molecular Sequence Data; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000502:physiology*; D011090:Polyenes / Q000494:pharmacology*; D012331:RNA, Fungal / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D014617:Vacuoles / Q000737:chemistry; D015003:Yeasts / Q000187:drug effects* / Q000235:genetics
chemical_list: D000935:Antifungal Agents; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D011090:Polyenes; D012331:RNA, Fungal; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); C083324:TOR1 protein, S cerevisiae; D019870:1-Phosphatidylinositol 4-Kinase; D020123:Sirolimus