Uncovering regulators of adaptor protein complex 1 (AP-1) trafficking pathways

Clathrin-coated vesicles (CCVs) traffic many cargo proteins throughout the cell to their functional locations. At the center of Golgi/endosome CCV transport is the heterotetrameric AP-1 adaptor protein complex, which coordinates cargo selection and vesicle formation. AP-1 is regulated by a suite of accessory proteins but their identity and functions are incompletely characterized. Here, we identified new AP-1 regulators through targeted yeast genome-wide screens performed in the Conibear lab. Adaptor protein complexes can contain variant subunits but the effect of this subunit exchange is unclear. In yeast, the functional relationship between the “classical” AP-1 complex containing the medium cargo-selective subunit Apm1 and the variant AP-1 complex (AP-1R) containing Apm2 is unclear. Our genome-wide screens indicated that they sort different cargo, and we found they also respond differently to small molecule inhibitors. We identified Mil1 as a novel specific regulator of the Apm2-containing complex, with active-site mutants supporting its role as a lipase. The data are consistent with a model where AP-1 and AP-1R are recruited to distinct membrane areas, facilitating different trafficking pathways. The second screen, for components involved in the trafficking of the AP-1 cargo Chs3, revealed a connection between the previously-identified AP-1 regulator Laa1 and an uncharacterized ORF that we named Laa2. Laa2 bridges AP-1 and Laa1 through an FGxF gamma-adaptin ear binding motif. Our identification of a yeast “Laa complex” consisting of Laa1, Laa2 and the short coiled-coil protein Slo1 led to the discovery of a similar complex in mammalian cells, consisting of HEATR5A, fasciculation and elongation protein zeta 2 (Fez2) and SCOC. We further showed that HEATR5A is distinct from HEATR5B, which works in the aftiphilin/γ-synergin complex previously implicated in AP-1 function. We found a conserved binding site in Laa2, Fez2, aftiphilin and the aftiphilin-related protein CLBA1 for HEATR5-family proteins, providing a new link between various trafficking pathways. This study identifies novel regulatory proteins that may facilitate AP-1 recruitment and function in particular pathways, and illustrates that proteins are not often purely redundant. Apm1 and Apm2 likely sort different cargo as part of distinct AP-1 isoforms, and HEATR5A and HEATR5B participate in distinct complexes.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat