Function and Regulation of Cargo Recognition by Clathrin Adaptors

University Of Colorado At Denver, Aurora CO

Investigators

Abstract

The orderly movement of membrane-enclosed compartments within the eukaryotic cell is critical to proper function. There are many different kinds of such intracellular membrane compartments, each with its own unique molecular mechanisms for biogenesis and intracellular placement and movement. A subset of such compartments utilizes a molecular mechanism for formation and trafficking that involves a protein called clathrin which forms a transient "coat" or "basket" around the membrane compartment. Such clathrin-coated membrane compartments are involved in the internalization of specific materials from the extracellular milieu (receptor-mediated endocytosis) and in the movement of newly synthesized proteins out to the cell surface. Thus, the population of "clathrin-coated vesicles" inside the cell is a heterogeneous mixture of compartments with different contents and therefore necessarily different destinations. This project concerns itself with understanding how the cell "knows" the content of such a compartment so that it "knows" where to send it. Clathrin adaptor protein complexes AP2 and AP1 are major components of clathrin coats at the plasma membrane and trans-Golgi network (TGN), respectively, where they participate in formation of clathrin-coated vesicles. These vesicles are responsible for the basic cellular functions of receptor-mediated endocytosis and organelle biogenesis. One of the key functions of AP complexes is to selectively recruit the integral membrane proteins ('cargo') transported by coated vesicles. The interaction of APs with cargoes is also implicated in the assembly of coats. In spite of a considerable amount of characterization of the biochemistry and cell biology of AP function, there are many aspects of their roles in membrane traffic that have yet to be defined. In particular, the role of AP-cargo interactions in their cellular function has only recently begun to be addressed through preliminary experiments that form the basis for this project. Several laboratories have established through in vitro experiments how APs recognize cargo by binding to sequence motifs (such as YxxQ, where x is any amino acid and Q is a bulky hydrophobic residue) in the cytoplasmic domains of receptors. Others have defined numerous regulatory proteins which interact with APs in cells. It is clear from studies of AP localization in cells that these molecules have very specific interactions with intracellular membranes at the sites of their function. It is not established, however, how much of their ability to function at different sites in the cell is determined by cargo recognition. Dr. Sorkin's lab has developed a unique system for examining the requirements for both AP2 and AP1 in protein sorting. In this system, they have successfully replaced the endogenous m1 and m2 subunits of the AP complexes by mutated versions of the same proteins. Specifically, they have mutated the cargo recognition site of the m2 subunit of AP2 and produced cells expressing the mutant AP2 complex, which allowed them to investigate the role of AP-cargo interactions in dictating AP localization and function. The expression of mutant m2 abolished the uptake of some plasma membrane receptors but do not affect the endocytosis of other cargoes. These data highlight the importance of a careful evaluation of the role of AP-cargo interaction in vivo. This project will extend these studies to a more in depth analysis of the effects of the m2 mutation on clathrin-coated pit formation and cargo targeting, and apply the same strategy to elucidate the role of m1 subunit in AP1 function. The comparative roles of m1 and m2 in different cellular locations will be defined by production of chimeric molecules between the two and analyzing membrane traffic in cells expressing these chimeras. The first objective is to test the hypothesis that interactions with YxxQ motifs, that are critical for cargo sorting, have negligible influence on targeting and docking of AP2 but is important for the assembly of AP1 coats. To this end, cells expressing mutant m2 or m1 subunit of AP2 or AP1, correspondingly, will be utilized to assess the role of m interactions with YxxQ-containing proteins in the correct targeting of APs and the assembly of coated pits/buds at the cell surface and TGN. The second objective is to test the hypothesis that AP1 plays a role in bi-directional TGN-endosomal trafficking through analysis of the effects of expression of mutant m1 subunits of AP1 incapable of YxxQ motif recognition. The comparative roles of m1 and m2 in different cellular locations will also be investigated in Objective 3 by production of chimeric molecules between the two and analyzing membrane traffic in cells expressing these chimeras. These studies will establish the role of AP-cargo interactions in directing membrane traffic and uncover new insights into regulatory mechanisms of protein sorting along the endocytic and secretory pathways.