Using unbiased and biophysical approaches to study clathrin coated vesicle formation

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AP-1 is a multimeric clathrin adaptor that is recruited to the trans Golgi Network (TGN) leading to cargo binding, clathrin coated vesicle formation and traffic of cargo to lysosomes or the plasma membrane (PM). The small GTPase Arf1 regulates the spatiotemporal recruitment of AP-1 and work from our group in live cells demonstrated that Arf1 also “opens” the core of AP-1, enabling efficient cargo binding. AP-1 is composed of two large subunits (1 and ), a medium subunit (µ1) and a small subunit (1). There are two isoforms of the µ1 subunit, µ1A which is ubiquitously expressed, and µ1B, whose expression is restricted to epithelial cells. Evidence points to a role for µ1B in TGN-to-PM trafficking, but the molecular mechanisms have not been well studied. In Aim 1, we will use BioID2 to map the interactome of µ1A and µ1B in live cells in both polarized and non-polarized states. BioID2 fuses a biotin ligase to a protein of interest (in this case µ1A and µ1B). Addition of biotin leads to efficient labeling of endogenous proteins within a short distance of the protein of interest. Mass spectrometry is then used to identify proteins that were in proximity with µ1A or µ1B following isolation by affinity chromatography using streptavidin beads (that bind biotinylated proteins). To further characterize the function of µ1A or µ1B, in Aim 2, we will use genome editing to knockout (KO) each of these genes. First, we will determine if KO of either of these genes affects their ability to polarize. Next, since AP-1 has an established role in sorting proteins to the lysosomal compartment and to the PM, we will determine changes to lysosomal and PM content using an unbiased approach in both µ1A-KO or µ1B-KO cells. Finally, in order to determine if µ1A or µ1B play a role in epithelial development, we will KO these proteins in adult liver stem cells and generate liver organoids (in collaboration with Dr. Daniel Cyr, INRS). Threonine 154 (T154) in µ1A is phosphorylated by the cyclin-G-associated Kinase (GAK), but the function of this post-translational modification (PTM) is unknown. Although µ1B also contains a threonine at this position, it is unknown if this site is also phosphorylated, and what the function of this PTM could be. In Aim 3, we will determine whether T154 in µ1B is phosphorylated and whether GAK also phosphorylates this site. Next, using BRET, we will determine if the phosphorylation site in µ1A and potential site in µ1B is required for membrane recruitment, interaction with Arf1 and whether it affects cargo interactions using well known cargo proteins and “hits” from our BioID2 screen. My research program aims to identify and characterize the proteins and mechanisms cells use to regulate intracellular trafficking at the TGN and endosomes. In the next 5 years, my goal is to understand the differences between µ1A and µ1B function in both non- and polarized cells and how PTMs can regulate these processes.