GRASP55 and GRASP65 play complementary and essential roles in Golgi cisternal stacking.
ABSTRACT: 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: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 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: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:Two classes of proteins that bind to each other and to Golgi membranes have been implicated in the adhesion of Golgi cisternae to each other to form their characteristic stacks: Golgi reassembly and stacking proteins 55 and 65 (GRASP55 and GRASP65) and Golgin of 45 kDa and Golgi matrix protein of 130 kDa. We report here that efficient stacking occurs in the absence of GRASP65/55 when either Golgin is overexpressed, as judged by quantitative electron microscopy. The Golgi stacks in these GRASP-deficient HeLa cells were normal both in morphology and in anterograde cargo transport. This suggests the simple hypothesis that the total amount of adhesive energy gluing cisternae dictates Golgi cisternal stacking, irrespective of which molecules mediate the adhesive process. In support of this hypothesis, we show that adding artificial adhesive energy between cisternae and mitochondria by dimerizing rapamycin-binding domain and FK506-binding protein domains that are attached to cisternal adhesive proteins allows mitochondria to invade the stack and even replace Golgi cisternae within a few hours. These results indicate that although Golgi stacking is a highly complicated process involving a large number of adhesive and regulatory proteins, the overriding principle of a Golgi stack assembly is likely to be quite simple. From this simplified perspective, we propose a model, based on cisternal adhesion and cisternal maturation as the two core principles, illustrating how the most ancient form of Golgi stacking might have occurred using only weak cisternal adhesive processes because of the differential between the rate of influx and outflux of membrane transport through the Golgi.
Project description:In vitro assays identified the Golgi peripheral protein GRASP65 as a Golgi stacking factor that links adjacent Golgi cisternae by forming mitotically regulated trans-oligomers. These conclusions, however, require further confirmation in the cell. In this study, we showed that the first 112 amino acids at the N-terminus (including the first PDZ domain, PDZ1) of the protein are sufficient for oligomerization. Systematic electron microscopic analysis showed that the expression of non-regulatable GRASP65 mutants in HeLa cells enhanced Golgi stacking in interphase and inhibited Golgi fragmentation during mitosis. Depletion of GRASP65 by small interference RNA (siRNA) reduced the number of cisternae in the Golgi stacks; this reduction was rescued by expressing exogenous GRASP65. These results provided evidence and a molecular mechanism by which GRASP65 stacks Golgi cisternal membranes. Further experiments revealed that inhibition of mitotic Golgi disassembly by expressing non-regulatable GRASP65 mutants did not affect equal partitioning of the Golgi membranes into the daughter cells. However, it delayed mitotic entry and suppressed cell growth; this effect was diminished by dispersing the Golgi apparatus with Brefeldin A treatment prior to mitosis, suggesting that Golgi disassembly at the onset of mitosis plays a role in cell cycle progression.
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:The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum, processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network. GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking and unconventional secretion. Previously, we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion or endoplasmic reticulum stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the trans-Golgi network.
Project description:GRASP65 and GRASP55 are peripheral Golgi proteins localized to cis and medial/trans cisternae, respectively. They are implicated in diverse aspects of protein transport and structure related to the Golgi complex, including the stacking of the Golgi stack and/or the linking of mammalian Golgi stacks into the Golgi ribbon. Using a mouse model, we interfered with GRASP65 by homologous recombination and confirmed its absence of expression. Surprisingly, the mice were healthy and fertile with no apparent defects in tissue, cellular or subcellular organization. Immortalized MEFs derived from the mice did not show any growth or morphological defects. However, despite the normal appearance of the Golgi ribbon, a fluorescence recovery after photobleaching assay revealed functional discontinuities specific to the cis cisternal membrane network. This leads to a strong change in the plasma membrane GSII lectin staining that was also observed in certain mutant tissues. These findings substantiate the role of GRASP65 in continuity of the cis Golgi network required for proper glycosylation, while showing that neither this continuity nor GRASP65 itself are essential for the viability of a complex organism.
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: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.