(1998) Neuron 20, 95C102 [PubMed] [Google Scholar] 45. revealed a different dysfunction at distal IS/OS regions that caused a slowly progressing retinal degeneration (35). Recent proteomic study has reported reduction in UNC119 expression as one of the possible culprits contributing to retinal degeneration in a mouse transgenic model (36). UNC119 was reported to interact with the N terminus of the GTP-bound Gt1 in Rabbit polyclonal to MICALL2 an acylation-dependent manner (28). Importantly, the return of Gt to the Incyclinide OS in the dark was impaired in knock-out mice (28). UNC119 was found to inhibit the GTPase activity of Gt1. Hence, the proposed model for transducin return to the OS in darkness is based on diffusion of the stable UNC119-Gt1GTP complex (28). Here, we examined the interaction of human UNC119 with transducin and the lipid specificity of UNC119 in comparison to that of PrBP/ in order to gain insights into the mechanism of ISOS transport of Gt in the dark. EXPERIMENTAL PROCEDURES Materials Myristoylated peptides, [MYR]-GAGASAEEK, and [MYR]-GCGASAEEK were synthesized by Proimmune Ltd. BC (3-(bromo acetyl)-7-diethyl amino coumarine) fluorophore was purchased from Molecular Probes, Inc. 6-((7-amino-4-methylcoumarin-3acetyl)amino)hexanoic acid, succinimidyl ester (AMCA-X, SE) was purchased from Invitrogen. (38). Gt1GDP was prepared and purified according to published protocol (39). Recombinant G11 were expressed using the baculovirus/sf9 cell system. The G1 baculoviral stock was obtained from Dr. S. Chen (University of Iowa). To generate G1 baculoviruses, the G1 cDNA was PCR amplified from bovine retinal library with the introduction of the coding sequence for the N-terminal His6 tag, and cloned into the pFast HTb vector using RsrII/NheI sites. Generation of the recombinant bacmids, transfection of Sf9 cells, and viral amplifications were carried out according to the manufacturer’s recommendations (Invitrogen). For expression of the G11 heterodimer, Sf9 cell cultures (2 106 cells/ml) were co-infected with G1 and G1 baculoviruses at MOI of 4C6. The G11 heterodimer was purified using affinity chromatography on Ni-NTA resin (Novagen) as previously described (40). The Gti chimera (Gt1*) with the His6 sequence inserted between Met115 and Pro116 of the helical domain of Ghi8 (41) was generated to allow the N-terminal myristoylation of the protein. The Gt11C115 sequence was PCR-amplified from the Chi8 template using a 5-primer with an NcoI site and a 3-primer coding the His6 sequence added to Gt1 specific sequence. The Chi8 116C350 sequence was PCR-amplified using a 5-primer with the His6-sequence added to the Gt1-specific sequence, and a 3-primer containing an XhoI site. The two resulting PCR products were used in the PCR reaction with the flanking primers, and the PCR product was subcloned into the pET15b vector using the NcoI/XhoI sites. Gt1* was expressed and purified as previously described (41). To obtain myristoylated Gt1* (myrGt1*), BL21-codon plus cells were co-transformed with kanamycin-resistant plasmid pbb131 expressing yeast cells was induced with the addition of 30 m IPTG. UNC119 and StrepII-UNC11955C240 were Incyclinide expressed overnight at 16 C, whereas UNC11955C240 and PrBP/ were expressed for 5 h at 30 C. The His6-tagged proteins were purified on Ni-NTA resin (Novagen). StrepII-UNC11955C240 was purified using StrepTactin Superflow Agarose (Novagen). UNC11955C240 and PrBP/ were additionally purified by gel-filtration on a Superdex 75 Sepharose column, and UNC119 was Incyclinide purified by an ion-exchange chromatography on a Uno-Q1 column (Bio-Rad). Preparation of Hypotonic, GTP, and GTPS Extracts of ROS Membranes To extract native transducin without.