Mammalian RAFT1 kinase domain provides rapamycin-sensitive TOR function in yeast.
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.
Alarcon
C M
CM
Department of Genetics, Duke University Medical Center, Durham, North Carolina 27710 USA.
Cardenas
M E
ME
Heitman
J
J
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
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