Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.Golgi biogenesis involves membrane tethering by two multifunctional proteins, GRASP65 and GRASP55, which are differentially localized to cis and medial Golgi cisternae, respectively. GRASP65 is associated with cis Golgi cisternae via both binding to the cis-localized golgin GM130 and insertion of its myristoylated N terminus (1, 2). GRASP55 is primarily on medial Golgi cisternae and binds medial-localized golgin-45 and other proteins and is myristoylated and palmitoylated (3, 4). Each protein contains a conserved N-terminal GRASP domain that mediates self-association, which results in the formation of homotypic tethering complexes that link analogous cisternae in adjacent ministacks (5-8).The GRASP region is predicted to contain two PDZ-like domains (4, 9). PDZ domains are ubiquitous globular proteinprotein interaction modules featuring a hydrophobic binding groove, which interacts with the C terminus of its target ligand, although recognition of internal sequences has also been observed (10, 11). Although recent work supports the presence of PDZ domains within the GRASP module (8), secondary structure predictions indicate significant mismatches to the typical organization of -strands and ␣-helices found in eukaryotic PDZs.Toward elucidating the structural mechanism of GRASPmediated tethering, we solved the crystal structure of the GRASP domain of GRASP55. Although the GRASP domain was indeed composed of two PDZ domains, the domains were circularly permuted, resulting in overall folds that were structurally more similar to prokaryotic PDZs. This unusual arrangement of a metazoan PDZ revealed that the key 2 strands of the binding grooves lay outside of the previously predicted PDZ-like regions. Significantly, an internal ligand sequence mapped in GRASP65 that binds to its first PDZ domain formed a surface projection that appeared to fit inside a deep depression within the PDZ1-binding pocket. Taken together, these data suggest a unique internal PDZ ligand interaction on opposite sides of the molecule and imply a multimeric tethering mechanism that mediates Golgi biogenesis. MATERIALS AND METHODSConstructs-GFP-ActA (8) was cloned into GRASP55 pCS-2 (12) using XbaI. To generate His-tagged GRASP55, GR...
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