Exomer complex

Abstract: Cargo adaptors control intracellular trafficking of transmembrane proteins by sorting them into membrane transport carriers. The COPI, COPII, and clathrin cargo adaptors are structurally well characterized, but other cargo adaptors remain poorly understood. Exomer is a specialized cargo adaptor that sorts specific proteins into trans-Golgi network (TGN)-derived vesicles in response to cellular signals. Exomer is recruited to the TGN by the Arf1 GTPase, a universally conserved trafficking regulator. Here, we report the crystal structure of a tetrameric exomer complex composed of two copies each of the Chs5 and Chs6 subunits. The structure reveals the FN3 and BRCT domains of Chs5, which together we refer to as the FBE domain (FN3–BRCT of exomer), project from the exomer core complex. The overall architecture of the FBE domain is reminiscent of the appendage domains of other cargo adaptors, although it exhibits a distinct topology. In contrast to appendage domains, which bind accessory factors, we show that the primary role of the FBE domain is to bind Arf1 for recruitment of exomer to membranes.

Abstract: The exomer complex is a putative vesicle coat required for the direct transport of a subset of cargoes from the trans-Golgi network (TGN) to the plasma membrane. Exomer comprises Chs5p and the ChAPs family of proteins (Chs6p, Bud7p, Bch1p, and Bch2p), which are believed to act as cargo receptors. In particular, Chs6p is required for the transport of the chitin synthase Chs3p to the bud neck. However, how the ChAPs associate with Chs5p and recognize cargo is not well understood. Using domain-switch chimeras of Chs6p and Bch2p, we show that four tetratricopeptide repeats (TPRs) are involved in interaction with Chs5p. Because these roles are conserved among the ChAPs, the TPRs are interchangeable among different ChAP proteins. In contrast, the N-terminal and the central parts of the ChAPs contribute to cargo specificity. Although the entire N-terminal domain of Chs6p is required for Chs3p export at all cell cycle stages, the central part seems to predominantly favor Chs3p export in small-budded cells. The cargo Chs3p probably also uses a complex motif for the interaction with Chs6, as the C-terminus of Chs3p interacts with Chs6p and is necessary, but not sufficient, for TGN export.

Abstract: Cells transport integral membrane proteins between organelles by sorting them into vesicles. Cargo adaptors act to recognize sorting signals in transmembrane cargos and to interact with coat complexes that aid in vesicle biogenesis. No coat proteins have yet been identified that generate secretory vesicles from the trans-Golgi network (TGN) to the plasma membrane, but the exomer complex has been identified as a cargo adaptor complex that mediates transport of several proteins in this pathway. Chs3, the most well-studied exomer cargo, cycles between the TGN and the plasma membrane in synchrony with the cell cycle, providing an opportunity to study regulation of proteins that cycle in response to signaling. Here we show that different segments of the Chs3 N-terminus mediate distinct trafficking steps. Residues 10-27, known to mediate retention, also appear to play a role in internalization. Residues 28-52 are involved in transport to the plasma membrane and recycling out of endosomes to prevent degradation in the vacuole. We also present the crystal structure of residues 10-27 bound to the exomer complex, suggesting different cargo adaptors could compete for binding to this segment, providing a potential mechanism for regulation.