| The presence of membrane bound compartments is a hallmark of eukaryotic cells. Much is known about trafficking between compartments within a cell. The mechanism of packaging cargo from the trans-Golgi network (TGN) into secretory vesicles destined for the plasma membrane (PM) is poorly understood. The exomer complex, consisting of a core protein, Chs5, and four paralogous proteins (Chs6, Bud7, Bch1, and Bch2) called the ChAPs (Chs5-Arf1-binding proteins) sorts and traffics a subset of cargo proteins at the TGN en route to the PM. It is the only cargo adaptor known to function in this pathway. The small GTPase Arf1, a master regulator of membrane trafficking, mediates exomer recruitment to the membrane surface. Here, I explore the structure and function of the exomer complex as it relates to complex formation, membrane recruitment, and membrane remodeling.;I determined the structure of a functional exomer complex consisting of Chs5-Chs6. I showed that the Chs5 subunit exhibits two distinct interactions with Chs6: one interaction mediates formation of the Chs5/Chs6 heterodimer, and the other mediates homodimerization of the Chs5/Chs6 heterodimer to form a tetramer. This structure revealed that the FN3 and BRCT (FBE) domains of Chs5 project away from the exomer core complex. The primary role of these domains is to bind Arf1 for recruitment of exomer to membranes. Providing additional affinity for the membrane is Chs6, which binds membranes independently of Arf1. Thus, multiple domains within exomer are required for localization to the TGN.;To better understand the molecular determinants of exomer recruitment, I determined the structure of an exomer-Arf1 complex. The structure revealed that exomer interacts with Arf1 through two interfaces. One interface involves a non-switch region of Arf1, binding in a novel nucleotide-independent manner to the FBE domain. Additionally, exomer interacts with Arf1 through the Arf1 switch regions. Furthermore, this structure allowed me to unambiguously identify the membrane-binding surface of the exomer complex. Surprisingly, Bch1 has a previously unrecognized hydrophobic membrane insertion motif that can cooperate with Arf1 to enhance membrane deformation and vesiculation. Thus, I have established a potential mechanism for the formation of secretory vesicles. |
