investigation; S. oleate medium for 18 h, subjected to nitrogen starvation for 2 h, and the subcellular localization of Atg36-GFP and Pex3-mCherry was observed under a fluorescence microscope. DIC, differential interference contrast microscopy. knockout cells. The phosphorylation of Atg36-GFP was observed in cells expressing WT Pex3-mCherry but was decreased in cells expressing Pex3-177CmCherry (Fig. 1was engineered to express Pex3-mCherry lacking the transmembrane domain name (Pex340C441-mCherry). In addition, because the protein level of Pex340C441-mCherry was lower than that of WT Pex3-mCherry, the promoter was replaced with the promoter to increase the protein level to that of WT Pex3-mCherry expressed under the original promoter (Fig. 1is important for Atg36 phosphorylation by Hrr25 and does not need to occur around the peroxisome. Pex3 directly promotes Atg36 phosphorylation by Hrr25 Our previous kinase assay using recombinant Hrr25 and GST-tagged Atg36 exhibited that Hrr25 directly phosphorylates Atg36 (24). The cytoplasmic region of Pex3 (Pex340C441) was purified and added to this reaction. When GST-Atg36 was incubated with Hrr25 and ATP in the absence of Pex340C441, the band of GST-Atg36 was slightly upshifted (Fig. 2knockout abolished this coprecipitation (Fig. 3and knockout cells with the proteasome inhibitor MG132 substantially increased Atg36-GFP in both oleate and nitrogen starvation media, suggesting that Atg36 is usually susceptible to degradation by the proteasome in the absence of Pex3 (Fig. 4cells (Fig. 4and was knocked out for efficient proteasome inhibition by MG132) were produced in oleate medium for 18 h. These cells were treated with or without MG132 in oleate medium or nitrogen starvation medium for 4 h and examined by immunoblotting using an anti-GFP antibody. analysis using recombinant proteins revealed that PI3K-gamma inhibitor 1 Pex3 directly promotes Atg36 phosphorylation by Hrr25 (Fig. 2). Moreover, co-immunoprecipitation analysis suggested that the conversation of Atg36 with Hrr25 is usually enhanced by Pex3 and that Hrr25 directly or indirectly interacts with Pex3 depending PI3K-gamma inhibitor 1 on Atg36 (Fig. 3). These results allow us to propose a model for the spatial regulation of Atg36 phosphorylation by Hrr25 (Fig. 5). Although cytoplasmic Atg36 is not a good substrate for Hrr25, it becomes efficiently phosphorylated by Hrr25 when bound to Pex3 in the peroxisomal membrane. Pex3 and Atg36 may both interact with Hrr25 to retain the kinase at the complex, facilitating Atg36 phosphorylation by Hrr25 (Fig. 5(formerly genes grew normally during this time period probably because SO+CA+ATU medium contained 0.1% glucose), and the medium was replaced with SD-N (0.17% YNB w/o aa and as and 2% glucose). 20 mm MG132 dissolved in DMSO was added to the media to a final concentration of 100 m for proteasome inhibition. Plasmids Oligonucleotides used for plasmid construction are listed in Table S2. The pGEX-6P-Pex340C441 plasmid for GST-Pex340C441 expression in was constructed as follows. The DNA sequence encoding Pex340C441 was amplified by PCR using genomic DNA from BY4741 (37) and the oligonucleotides Pex340C441 forward and reverse. Products were cloned into the pGEX-6P-1 vector (GE Healthcare) using BamHI and EcoRI. The mutation Rabbit Polyclonal to OR52D1 (F64S,T74A,H354L) (19) was introduced into this plasmid using the QuikChange Site-Directed Mutagenesis Kit (Agilent) in two actions with the pairs of oligonucleotides Pex3 F64ST74A forward and reverse and Pex3 H354L forward and reverse, resulting in pGEX-6P-Pex3-17740C441. pGEX-6P-Atg36 and pGEX-6P-Hrr25 have been described previously (24). To construct the plasmid expressing Pex3-2mCherry (pRS316-sequence with the promoter and the terminator was amplified by PCR using genomic DNA from YMS28 and the oligonucleotides Pex3-2mCherry 500 bp up and Pex3-2mCherry-PGKter down, and cloned into the single-copy vector pRS316 using BamHI and EcoRI. The mutation was introduced into this plasmid as described above to construct pRS316-for 5 min at 4C. The pellets were suspended in (5 for 1 min, and the supernatants were used for immunoblotting. Monoclonal antibodies against GFP (JL-8; Clontech), mCherry (a gift from Dr. Toshiya Endo), GST (B-14; Santa Cruz Biotechnology), and myc (9E10; Santa Cruz Biotechnology) were used for the detection of tagged PI3K-gamma inhibitor 1 proteins. Fluorescence microscopy Fluorescence microscopy was performed as described previously (38) using an inverted fluorescence microscope (IX83; Olympus) equipped with an electron-multiplying charged coupled device camera (ImagEM C9100-13; Hamamatsu Photonics), a 150 objective lens (UAPON 150XOTIRF, NA/1.45; Olympus), a 488 nm blue.