Hill K

Hill K., Li Y., Bennett M., McKay M., Zhu X., Shern J., Torre E., Lah J. Golgi by mutating key residues within them and examining adaptor recruitment at the Golgi and traffic to post-Golgi site(s). We demonstrate strict specificity for recruitment of the Mint3 adaptor by APP at the Golgi, a critical role for Tyr-682 (within the YENPTY motif) in Mint3 recruitment and export of APP from the Golgi, and we identify LAMP1+ structures as the proximal destination of APP after leaving the Golgi. Together, these data provide a detailed view of the first sorting step in its route to the cell surface and processing by secretases and further highlight the critical role played by Mint3. is Lafutidine any amino acid and is a hydrophobic one) adaptin-binding motif (653YTSI656) functions in the basolateral sorting of APP in at least some cell types (21, 22), as well as in internalization at the plasma membrane (23). The more membrane-distal portion of the tail contains a Yschematic representation of APP, consisting of the lumenal, transmembrane (indicates that the lumenal and transmembrane domains of APP were replaced with those of CD8. HeLaM cells were transfected with the cargos indicated and 18 h later were fixed and stained with antibodies against APP and Mint3 (and Mint3 recruitment was quantified in cells treated as described above. Stacks of wide field images were collected and deconvolved, and isosurfaces were generated based on APP staining ( 7 cells were used per condition. ANOVA was use to compare groups to mock-transfected cells (indicate statistical significance compared with mock-transfected ( 0.05). total cellular Mint3 is unaffected by expression of APP mutants. HeLaM cells were transfected with the CD8-APP (if APP has more than a single means for exit from the Golgi the nature of the adaptor(s) it recruits will likely result in different routes to the plasma membrane. This has the potential for delivering APP to different sites for processing, resulting in differences in the location and extent of A generation. Clearly, a better understanding of the regulation of export of APP from the Golgi will provide not only a better understanding of the regulation of this process but may also provide targets for clinically relevant intervention. Previous work from our laboratory demonstrated that APP recruits Mint3 for export from the Golgi, and a truncation of the cytoplasmic tail of APP to eliminate the YENPTY motif eliminated Mint3 recruitment and altered APP export (25). However, this truncation also eliminated other known adaptor-binding motifs, most importantly to our interpretation is the motif that binds to AP-4. Thus, we Lafutidine wanted to refine these studies Lafutidine using site-directed mutagenesis to determine the impact on binding and functionality of a much more discrete number of binding partners. To evaluate the effect of the sorting motifs on the export of APP from the Golgi and proximal destination, we mutated key residues within the cytosolic tail of APP and evaluated effects Lafutidine on adaptor recruitment at the Golgi and traffic to post-Golgi sites. We also took advantage Lafutidine of previously described protocols that can arrest protein export from the Golgi and accumulate a bolus of cargo that is more easily tracked (20 C temperature block) or strip the Golgi of Arf-dependent adaptors (short term exposure to the drug brefeldin A (BFA)) to ask specific questions about the impact of specific residues in the cytoplasmic tail of APP on adaptor recruitment and Golgi export and proximal destination. EXPERIMENTAL PROCEDURES Cell Culture HeLaM (generous gift from Dr. Margaret Robinson) cells were maintained in 10% fetal bovine serum (Atlanta Biologicals, catalog no. “type”:”entrez-protein”,”attrs”:”text”:”S11150″,”term_id”:”98016″,”term_text”:”pirS11150) in DMEM (Invitrogen catalog no. 11965, v/v) in a water-jacketed incubator and maintained at 5% CO2 and 37 C. Plasmids and Transfections Generation of the CD8-tail constructs is described by Caster (33). Each of Rabbit polyclonal to EpCAM these constructs expresses the ectodomain and.