2001;21:3564C3575. these membranous sites. On the other hand, that Kog1 is available by us, the candida homologue from the mammalian Tor regulatory proteins Raptor, interacts with Tor1p preferentially. These findings offer proof for the lifestyle of Tor signaling complexes which contain distinct as well as overlapping components. That these complexes colocalize to a membrane-bound compartment suggests an intimate relationship between membrane-mediated signaling and Tor activity. INTRODUCTION Understanding how cell growth is controlled in response to environmental signals remains an outstanding biological problem. It has become clear in recent years that the Tor kinases act within an intracellular regulatory network used by eukaryotic cells to regulate their growth according to nutrient availability (reviewed by Dennis and genes (Heitman target genes, that encode mitochondrial and peroxisomal enzymes required for de novo biosynthesis of glutamate and glutamine (Komeili MATa). Culture medium Idazoxan Hydrochloride used was YPD (2% yeast extract, 1% peptone, and 2% dextrose). Yeast cultures were grown at 30C for all experiments. DNA ligations were performed using the Rapid DNA ligation kit (Roche Diagnostics, Indianapolis, IN). Plasmid DNA constructs were transformed into DH5 and grown at 37C. Yeast transformations were performed using a lithium acetate procedure (Geitz and Woods, 2002 ). Synthetic complete dextrose medium (0.8% yeast nitrogen base without amino acids, pH 5.5, 2% dextrose) was supplemented with amino acids as described previously (Sherman, 1991 ). Rapamycin (Sigma-Aldrich, St. Louis, MO) was dissolved in dimethyl Idazoxan Hydrochloride sulfoxide (DMSO) and added to a final concentration of 0.2 g/ml. Anti-hemagglutinin (HA) (HA.11) and anti-Myc (9E10) monoclonal antibodies were purchased from Covance (Berkeley, CA). Anti-Tor1p polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Plasmid Construction Plasmid pFA6a-HisMX6-PTOR2-ATG-3HA Idazoxan Hydrochloride (Figure ?(Figure1A),1A), used for amino terminal tagging of with three copies of the HA epitope (HA3) by integration into the genome of W303a, was constructed in several steps. First, plasmid pFA6a-His3MX6-PGAL1C3HA (Brachmann (600 base pairs) directly upstream of the start was polymerase chain reaction (PCR) amplified by using primers that contained the was the same as described above except for the following: the pFA6a-HisMX6-PTOR2-start-3HA plasmid was digested with promoter. The promoter regions of (240 base pairs) directly upstream of the start codon was amplified using genomic DNA from strain S288c as a template. Herein, the forward primer included a within the genome, PCR was performed using pFA6a-HisMX6-PTOR2-start-3HA and two gene was disrupted in the HA3-TOR2 strain by using a similar PCR-based method and plasmid pFA6a-TRP1 as template (Longtine open reading frame. To verify that strain HA3-TOR2 produced full-length HA3-Tor2p protein, Western blot analysis was performed. Briefly, 5 OD of cells from strain HA3-TOR2 W303a was pelleted and resuspended in 0.5 ml of trichloroacetic acid (TCA) buffer plus protease and phosphatase inhibitors. Resuspended cells were combined with 0.5 mg of glass beads and 0.5 ml of cold 30% TCA and were vortexed for four 30-s IL2RG intervals with cooling on ice during the interim. Supernatants were centrifuged at 20,000 for 5 min at 4C. Pellets were washed with 1 ml of cold acetone and centrifuged at 20,000 for 5 min at 4C. Acetone was completely removed and pellets were air dried for 10 min. Pellets were resuspended in sample buffer, incubated for 5C10 min at 65C, and loaded onto 7.5% SDS-PAGE gels followed by staining with Coomassie Blue or transfer to nitrocellulose for analysis for Western blotting. Anti-HA monoclonal antibody was used to detect HA3-Tor2p. A similar approach was used to confirm the sizes of the other epitope-tagged proteins described above. Antibody Production Anti-HA polyclonal antibodies were raised by immunizing rabbits with a peptide sequence from the influenza virus HA epitope (peptide sequence CYPYDVPDYA) conjugated to keyhole limpet hemocyanin. HA antibodies were affinity purified from serum by using a purified glutathione for 20 min at 4C. The supernatants (S1) were pooled and centrifuged again as described above..