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A Membrane Trafficking Screen To Identify Components Involved In Clathrin‐Independent Endocytosis

Endocytosis is an important event that cells utilize to internalize cell surface proteins and fluid into the cell. There are two main forms of endocytosis: clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE), amongst which CME is most studied. While CME is important for the internalization of many surface receptors, bulk trafficking of membrane and fluid occurs primarily in a clathrin‐independent manner. Our lab has focused on CIE associated with Arf6, although other CIE pathways exist for specific cargoes and in different cell types. While we have a fair understanding of the trafficking and sorting of the cargoes that utilize CIE, we lack understanding of the entry mechanism. We know that CIE occurs independently of clathrin, the clathrin adaptor protein (AP2), and dynamin. Among the types of CIE cargo proteins, we have focused on two ubiquitously expressed proteins, the major histocompatibility complex class I (MHCI) and CD59, a GPI‐anchored protein, with an objective to identify requirements for their entry. Therefore, we performed an siRNA screen using the Dharmacon™ Membrane Trafficking library, which targets 140 genes, to identify proteins essential for the internalization of CD59 and MHCI in Hela cells. Target genes were individually knocked down and cells were allowed to internalize CIE cargo proteins. Following internalization, the proteins were visualized by immunofluorescence and the amount of internalized protein was quantified by image analysis software. Depletion of each candidate gene was scored as either inhibiting or enhancing internalization based on the relative amount of internalization compared to that observed with the non‐target siRNA control cells. The knocked‐down genes that lead to inhibition of internalization might represent components involved in CIE entry and the knocked‐down genes that lead to “enhanced” internalization could be negative regulators of this process. The hits from the initial screen were followed up by other confirmatory tests with individual siRNA oligo sequences. Interestingly, different proteins appeared as hits for MHCI and CD59 suggesting the differential regulation of integral membrane proteins vs. GPI‐anchored proteins in CIE. Surprisingly, among them are distinct subunits of AP2 that inhibited MHCI internalization specifically, but not CD59. Knocking down individual alpha or beta subunits of AP2 inhibited MHCI internalization whereas knockdown of the mu subunit, which is typically knocked down to demonstrate a requirement for AP2/clathrin, had no such effect. This raises the question of whether components of AP2 can function in CIE entry independent of the involvement of CME. We hope to understand the function of these two AP2 subunits in CIE in future studies.