Using unbiased and biophysical approaches to study clathrin coated vesicle formation

AP-1是一种多聚体网格蛋白衔接子，其被募集到反式高尔基体网络（TGN），导致货物结合、网格蛋白包被的囊泡形成和货物向溶酶体或质膜（PM）的运输。小的GTd-Arf 1调节AP-1的时空募集，我们小组在活细胞中的工作表明，Arf 1还“打开”AP-1的核心，使有效的货物结合成为可能。 AP-1由两个大亚基（β 1和β 2）组成，一个中亚基（µ1）和一个小亚基（s1）。µ1亚基有两种亚型，µ1A广泛表达，µ1B仅限于上皮细胞表达。有证据表明，µ1B在TGN到PM的运输中发挥作用，但分子机制尚未得到充分研究。在目标1中，我们将使用BioID 2来绘制极化和非极化状态下活细胞中µ1A和µ1B的相互作用组。BioID 2将生物素连接酶与目标蛋白质（在本例中为µ1A和µ1B）融合。生物素的添加导致在目标蛋白质的短距离内有效标记内源性蛋白质。然后使用质谱法鉴定在使用链霉亲和素珠（其结合生物素化蛋白质）通过亲和色谱分离后与µ1A或µ1B接近的蛋白质。 为了进一步表征µ1A或µ1B的功能，在目标2中，我们将使用基因组编辑来敲除（KO）这些基因中的每一个。首先，我们将确定这些基因中的任何一个的KO是否会影响它们的能力。接下来，由于AP-1在将蛋白分选到溶酶体区室和PM中具有既定作用，因此我们将在µ1A-KO或µ1B-KO细胞中使用无偏方法确定溶酶体和PM含量的变化。最后，为了确定µ1A或µ1B是否在上皮发育中发挥作用，我们将在成人肝脏干细胞中KO这些蛋白质并产生肝脏类器官（与INRS的丹尼尔Cyr博士合作）。 µ1A中的T154被细胞周期蛋白G相关激酶（GAK）磷酸化，但这种翻译后修饰（PTM）的功能尚不清楚。虽然µ1B在这个位置也含有一个苏氨酸，但不知道这个位点是否也被磷酸化，以及这个PTM的功能是什么。在目标3中，我们将确定µ1B中的T154是否被磷酸化，以及GAK是否也磷酸化该位点。接下来，使用BRET，我们将确定μ1A中的磷酸化位点和μ1B中的潜在位点是否是膜募集、与Arf 1相互作用所必需的，以及它是否影响使用众所周知的货物蛋白和来自我们BioID 2筛选的“命中”的货物相互作用。 我的研究项目旨在识别和表征细胞用于调节TGN和内体细胞内运输的蛋白质和机制。在接下来的5年里，我的目标是了解非极化细胞和极化细胞中µ1A和µ1B功能的差异，以及PTM如何调节这些过程。

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 (ß1 and ?), a medium subunit (µ1) and a small subunit (s1). 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.