The long term goal of this research is to understand the molecular events responsible for the movement of receptors between intracellular compartments. Receptor trafficking is critical to normal cell growth because delivery of receptor-ligand complexes to endosomes is a first step in turning off growth factor signaling. One such important receptor is the mannose 6-phosphate receptor (MPR), which delivers newly synthesized lysosomal enzymes from the trans-Golgi network (TGN) to late endosomes, and then recycles back to the TGN to repeat the cycle. Like all receptor transport events, this process requires the cooperative effort of many types of proteins, including Rab GTPases, tethers, and SNARE proteins. MPR recycling requires Arl1, Rab6, and Rab9 GTPases, and Rab9 effectors p40, T1P47, GCC185, and RhoBTB3. Depletion of these factors using small interfering RNA (siRNA) causes disruption of normal juxta-nuclear MPR localization, and results in accumulation of MPR vesicles at the cell periphery (""""""""MPR dispersal""""""""). Detection of the MPR dispersal phenotype can be automated computationally. This proposal aims to identify novel proteins that participate in MPR trafficking by 1) performing a genome-wide siRNA screen, selecting for siRNAs that yield an MPR dispersal phenotype, 2) distinguishing siRNAs that block receptor endocytosis from those that block transport to the Golgi, and 3) determining whether candidate siRNAs that block transport to the Golgi are blocked at retrieval from the early endosome or the late endosome. The screen will be perfomed by transfecting cultured epithelial cells with 21,122 siRNA pools in triplicate in 348-well clear-bottomed plates. After 72 hrs, the cells will be fixed, stained with antibodies against MPRs and the Golgi, imaged with a high throughput microscope, and analyzed for MPR dispersal with custom image analysis software. Secondary screens will allow identification of the specific, altered trafficking step, and interesting candidates will be further characterized. This approach should yield an important list of proteins that function in promoting vesicle formation, motility, docking, and fusion. Though the ability to move receptors between compartments in a cell is important for normal cellular growth control, only some of the proteins that regulate this process are known. This proposal seeks to identify novel human proteins that participate in receptor trafficking by performing a large-scale genetic screen using molecules called small interfering RNAs. Defining the molecular basis of receptor transport pathways will provide information crucial to understanding the control of normal cellular growth.
|Lee, Peter L; Ohlson, Maikke B; Pfeffer, Suzanne R (2015) Rab6 regulation of the kinesin family KIF1C motor domain contributes to Golgi tethering. Elife 4:|