Identification and Characterization of Regulators of GLUT4 Trafficking

Cargo proteins moving between organelles are transported by membrane-enclosed vesicles. Identifying the factors regulating vesicle-mediated transport remains a major challenge in mammalian cells. Here, we performed unbiased genome-wide CRISPR-Cas9 genetic screens to systematically dissect insulin-dependent translocation of glucose transporters (GLUTs), a classic vesicle transport pathway crucial to mammalian physiology. These screens identified known regulators of the pathway as well as a large number of unknown regulatory factors that we validated in secondary screens. The identified genes encode established or predicted factors involved in vesicle budding or fusion, cargo sorting, signal transduction, cell motility, and cellular metabolism, as well as proteins lacking annotated functions. Mechanistic analysis demonstrated that Rab-interacting factor (RABIF), a putative guanine nucleotide exchange factor (GEF), positively regulates GLUT translocation by stabilizing Rab GTPases, a new function independent of its GEF catalytic activity. Alpha- and gamma-adaptin binding protein (AAGAB), identified as a negative regulator of GLUT translocation, acts as a key regulator of AP2 adaptor formation in clathrin-mediated endocytosis. Mass spectrometry-based proteomic analysis showed that both AAGAB and RABIF regulate diverse cargo proteins, suggesting that the factors identified in our screens play broad roles in vesicle transport regulation. Our findings reveal new facets of vesicle-mediated cargo transport and suggest a general strategy for genetically dissecting complex membrane processes in mammalian cells