Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis.
ABSTRACT: 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.
Project description:The stacking of Golgi cisternae involves GRASP65 and GRASP55. The oligomerization of the N-terminal GRASP domain of these proteins, which consists of two tandem PDZ domains, is required to tether the Golgi membranes. However, the molecular basis for GRASP assembly is unclear. Here, we determined the crystal structures of the GRASP domain of GRASP65 and GRASP55. The structures reveal similar homotypic interactions: the GRASP domain forms a dimer in which the peptide-binding pockets of the two neighboring PDZ2 domains face each other, and the dimers are further connected by the C-terminal tail of one GRASP domain inserting into the binding pocket of the PDZ1 domain in another dimer. Biochemical analysis suggests that both types of contacts are relatively weak but are needed in combination for GRASP-mediated Golgi stacking. Our results unveil a novel mode of membrane tethering by GRASP proteins and provide insight into the mechanism of Golgi stacking.
Project description:Golgin45 is required for normal Golgi structure and the transportation of protein from the ER. It forms a specific complex with GRASP55 in vivo Little is known regarding the molecular details of this interaction and its structural role in stacking of the Golgi complex. Here, we present the crystal structure of the GRASP domains of GRASP55 in complex with the Golgin45 C-terminal peptide, determined at 1.33 Å resolution. Similar to the structure of GRASP65 bound to GM130 reported recently, this structure reveals more than one interacting site and involves both PDZ1 and PDZ2 domains of the GRASP simultaneously. The C-terminal peptides of Golgin45 and GM130 present a conserved PDZ domain binding motif sequence and recognize the canonical PDZ-peptide binding groove of the PDZ1 domains of GRASP55 and GRASP65. A main difference in this recognition process resides in a structural rearrangement of GRASP65-GM130 that does not occur for the GRASP55-Golgin45 complex. The binding site at the cleft between the PDZ1 and PDZ2 domains of GRASP65 is dominated by hydrophobic interactions with GM130 that are not observed in the GRASP55-Golgin45 complex. In addition, a unique zinc finger structure is revealed in the GRASP55-Golgin45 complex crystal structure. Mutagenesis experiments support these structural observations and demonstrate that two of these sites are required to form a stable complex. Finally, a novel Golgi stacking model is proposed according to these structural findings.
Project description:GM130 and GRASP65 are Golgi peripheral membrane proteins that play a key role in Golgi stacking and vesicle tethering. However, the molecular details of their interaction and their structural role as a functional unit remain unclear. Here, we present the crystal structure of the PDZ domains of GRASP65 in complex with the GM130 C-terminal peptide at 1.96-Å resolution. In contrast to previous findings proposing that GM130 interacts with GRASP65 at the PDZ2 domain only, our crystal structure of the complex indicates that GM130 binds to GRASP65 at two distinct sites concurrently and that both the PDZ1 and PDZ2 domains of GRASP65 participate in this molecular interaction. Mutagenesis experiments support these structural observations and demonstrate that they are required for GRASP65-GM130 association.
Project description:Homotypic membrane tethering by the Golgi reassembly and stacking proteins (GRASPs) is required for the lateral linkage of mammalian Golgi ministacks into a ribbon-like membrane network. Although GRASP65 and GRASP55 are specifically localized to cis and medial/trans cisternae, respectively, it is unknown whether each GRASP mediates cisternae-specific tethering and whether such specificity is necessary for Golgi compartmentalization. Here each GRASP was tagged with KillerRed (KR), expressed in HeLa cells, and inhibited by 1-min exposure to light. Significantly, inactivation of either GRASP unlinked the Golgi ribbon, and the immediate effect of GRASP65-KR inactivation was a loss of cis- rather than trans-Golgi integrity, whereas inactivation of GRASP55-KR first affected the trans- and not the cis-Golgi. Thus each GRASP appears to play a direct and cisternae-specific role in linking ministacks into a continuous membrane network. To test the consequence of loss of cisternae-specific tethering, we generated Golgi membranes with a single GRASP on all cisternae. Remarkably, the membranes exhibited the full connectivity of wild-type Golgi ribbons but were decompartmentalized and defective in glycan processing. Thus the GRASP isoforms specifically link analogous cisternae to ensure Golgi compartmentalization and proper processing.
Project description:We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.
Project description:GRASP65 and GRASP55 were classified as Golgi reassembly stacking proteins which play crucial and complementary roles in the stacking of Golgi cisternae. They also participate in vesicle tethering, mitotic progression, the disassembly and reassembly of the Golgi apparatus during mitosis and unconventional secretory pathway regulation. In this study, the expression, crystallization and preliminary crystallographic analysis of the GRASP65 GRASP domain from Rattus norvegicus are presented. The crystals diffracted to 2.0 Å resolution and belonged to space group P21212, with unit-cell parameters a = 44.99, b = 104.29, c = 37.93 Å, ? = ? = ? = 90°. Furthermore, molecular replacement was employed to determine the structure of the GRASP65 GRASP domain from R. norvegicus.
Project description:In vitro studies have suggested that Golgi stack formation involves two homologous peripheral Golgi proteins, GRASP65 and GRASP55, which localize to the cis and medial-trans cisternae, respectively. However, no mechanism has been provided on how these two GRASP proteins work together to stack Golgi cisternae. Here, we show that depletion of either GRASP55 or GRASP65 by siRNA reduces the number of cisternae per Golgi stack, whereas simultaneous knockdown of both GRASP proteins leads to disassembly of the entire stack. GRASP55 stacks Golgi membranes by forming oligomers through its N-terminal GRASP domain. This process is regulated by phosphorylation within the C-terminal serine/proline-rich domain. Expression of nonphosphorylatable GRASP55 mutants enhances Golgi stacking in interphase cells and inhibits Golgi disassembly during mitosis. These results demonstrate that GRASP55 and GRASP65 stack mammalian Golgi cisternae via a common mechanism.
Project description:Transforming growth factor-alpha (TGF-alpha) and related proteins represent a family of transmembrane growth factors with representatives in flies and worms. Little is known about the transport of TGF-alpha and other transmembrane growth factors to the cell surface and its regulation. p59 was purified as a cytoplasmic protein, which at endogenous levels associates with transmembrane TGF-alpha. cDNA cloning of p59 revealed a 452 amino acid sequence with two PDZ domains. p59 is myristoylated and palmitoylated, and associates with the Golgi system, where it co-localizes with TGF-alpha. Its first PDZ domain interacts with the C-terminus of transmembrane TGF-alpha and select transmembrane proteins. p59 is the human homolog of GRASP55, which is structurally related to GRASP65. GRASP55 and GRASP65 have been shown to play a role in stacking of the Golgi cisternae in vitro. C-terminal mutations of transmembrane TGF-alpha, which decrease or abolish the interaction with p59, also strongly impair cell surface expression of TGF-alpha. Our observations suggest a role for membrane tethering of p59/GRASP55 to select transmembrane proteins, including TGF-alpha, in maturation and transport to the cell surface.
Project description:Golgi reassembly stacking protein of 65 kDa (GRASP65) and Golgi reassembly stacking protein of 55 kDa (GRASP55) were originally identified as Golgi stacking proteins; however, subsequent GRASP knockdown experiments yielded inconsistent results with respect to the Golgi structure, indicating a limitation of RNAi-based depletion. In this study, we have applied the recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology to knock out GRASP55 and GRASP65, individually or in combination, in HeLa and HEK293 cells. We show that double knockout of GRASP proteins disperses the Golgi stack into single cisternae and tubulovesicular structures, accelerates protein trafficking, and impairs accurate glycosylation of proteins and lipids. These results demonstrate a critical role for GRASPs in maintaining the stacked structure of the Golgi, which is required for accurate posttranslational modifications in the Golgi. Additionally, the GRASP knockout cell lines developed in this study will be useful tools for studying the role of GRASP proteins in other important cellular processes.
Project description:The multi-domain scaffolding protein NHERF1 modulates the assembly and intracellular trafficking of various transmembrane receptors and ion-transport proteins. The two PDZ (postsynaptic density 95/disk large/zonula occluden 1) domains of NHERF1 possess very different ligand-binding capabilities: PDZ1 recognizes a variety of membrane proteins with high affinity, while PDZ2 only binds limited number of target proteins. Here using NMR, we have determined the structural and dynamic mechanisms that differentiate the binding affinities of the two PDZ domains, for the type 1 PDZ-binding motif (QDTRL) in the carboxyl terminus of cystic fibrosis transmembrane regulator. Similar to PDZ2, we have identified a helix-loop-helix subdomain coupled to the canonical PDZ1 domain. The extended PDZ1 domain is highly flexible with correlated backbone motions on fast and slow timescales, while the extended PDZ2 domain is relatively rigid. The malleability of the extended PDZ1 structure facilitates the transmission of conformational changes at the ligand-binding site to the remote helix-loop-helix extension. By contrast, ligand binding has only modest effects on the conformation and dynamics of the extended PDZ2 domain. The study shows that ligand-induced structural and dynamic changes coupled with sequence variation at the putative PDZ binding site dictate ligand selectivity and binding affinity of the two PDZ domains of NHERF1.