The Golgi complex is a central membrane organelle for the trafficking and processing of membrane and secretory proteins in all eukaryotic cells. A unique feature of the Golgi structure is a stack of flattened cisternae. However, how this structure is generated is poorly understood. Studying the biogenesis and molecular organization of this organelle is fundamental to understanding the mechanism of protein trafficking under normal and disease conditions. We use a multidisciplinary approach to determine the mechanism and significance of Golgi stacking and the role of ubiquitin in Golgi biogenesis in the mammalian cell cycle. We have also gathered intriguing preliminary data concerning Golgi defects in human diseases such as cancer and Alzheimer’s disease.

Fig 1. Golgi cycle-1a

Fig. 1. Golgi disassembly and reassembly in the cell cycle. A. Fluorescence images of Golgi disassembly and reassembly in the cell cycle. A GFP-tagged Golgi enzyme was used as a marker. Bar: 5 µm. B. EM images of in vitro reconstituted Golgi disassembly and reassembly in the cell cycle. Bar: 0.5 µm.

The approach: the in vitro Golgi disassembly and reassembly assay and Golgi systems biology
We have developed a multidisciplinary approach employing biochemistry, cell biology, electron microscopy, proteomics and glycomics, combined with a novel in vitro reconstitution assay, to exploit the mechanism of Golgi structure formation and function. In mammalian cells, biogenesis of the Golgi occurs through a unique disassembly and reassembly process during each cycle of cell division. The Golgi is fragmented at the onset of mitosis, dispersing stacks that further vesiculate, yielding thousands of vesicles distributed throughout the cytoplasm. In telophase, Golgi vesicles are divided equally between the daughter cells, where they are reassembled into stacks and ribbons (Fig. 1A). We have developed an in vitro system that replicates the Golgi disassembly and reassembly process through treatment of purified Golgi membranes with mitotic (MC) or interphase (IC) cytosol prepared from spinner HeLa cells (Fig. 1B), or with purified proteins. The use of purified proteins in this assay makes it amenable to biochemical manipulation and thus provides a powerful tool for testing key proteins in Golgi structure formation. This allowed us to identify the minimal machinery and key components that control mitotic Golgi disassembly and post-mitotic reassembly. Mitotic disassembly is mediated by cisternal unstacking and vesiculation; post-mitotic Golgi reassembly involves cisterna formation by membrane fusion and Golgi stacking (more details below).

Fig 2. Stacking model

Fig. 2. Model for the role of GRASP65 in Golgi stacking. During interphase, GRASP65 dimers from adjacent cisternal membranes oligomerize to form a “glue” to hold the membranes together. During mitosis, mitotic phosphorylation of GRASP65 breaks the trans-oligomers and unstacks the cisternae. Postmitotic dephosphorylation of GRASP65 by PP2A leads to re-oligomerization.

Fig 3. Stacking_trafficking_glyco

Fig. 3. Golgi destruction accelerates protein trafficking and impairs accurate glycosylation. When Golgi cisternae are fully stacked (A), vesicles can only form and fuse at the rims. This slows down trafficking but enforces accurate glycosylation. Once the cisternae are unstacked (B), more membrane area becomes accessible for vesicle budding and fusion, thereby increasing cargo transport. This, however, causes glycosylation and sorting defects.

Golgi stacking: how and why Golgi cisternae form stacks?
We study the Golgi stacking proteins GRASP55 and GRASP65 in Golgi stack formation. A characteristic structural feature of the Golgi apparatus is the formation of Golgi stacks. However, the significance of cisternal stacking for normal cell physiology remains largely untested because, until recently, there were no suitable molecular tools to control stack formation. Our recent work demonstrated that GRASP55 and GRASP65 function as the “glue” that sticks the cisternae together into stacks by forming mitotically regulated trans-oligomers (Fig. 2). Antibodies against GRASP55 or GRASP65 inhibited post-mitotic stacking of newly formed cisternae both in the in vitro Golgi disassembly and reassembly assay and in cells. Expression of non-phosphorylatable mutants of GRASP55/65 increased the number of cisternae per stack. Depletion of either GRASP55 or GRASP65 by RNA interference (RNAi) reduced the number of cisternae per stack, while simultaneous depletion of both GRASPs resulted in complete disassembly of the Golgi. We are currently characterizing GRASP55 and GRASP65 and their interacting proteins in details to understand how this unique structure is organized at the molecular level.

The identification of GRASP55/65 also allows us to manipulate Golgi stack formation and thus to determine the biological significance of Golgi structure formation. Golgi fragmentation has major effects on protein transport and processing. First, Golgi destruction accelerates protein trafficking. Inhibition of Golgi stack formation by microinjected GRASP65 antibodies accelerates CD8 trafficking. Depletion of both GRASPs destroys the Golgi structure and enhances trafficking of the cell adhesion protein integrin, the vesicular stomatitis virus G glycoprotein (VSVG), and the lysosomal enzyme cathepsin D. The Golgi tethering protein GM130 is unaffected, indicating that the observed effect is not caused by the disruption of membrane tethering. Golgi destruction also increases the rate and efficiency of COPI vesicle formation in vitro and membrane association of coat proteins in cells. Second, Golgi destruction impairs accurate protein glycosylation. GRASP depletion does not impact the expression level and localization of Golgi enzymes, but decreases sialic acid levels on the cell surface. Third, Golgi destruction causes missorting of proteins, e.g. cathepsin D precursor, to the extracellular space. We propose that Golgi stack formation is a flux regulator for protein trafficking and thereby functions as a quality control mechanism for protein sorting and modifications, especially glycosylation. (Fig. 3).

Fig 4. DisA-ReA-Ub Model3a

Fig. 4. Schematic illustration of the mechanism of Golgi disassembly and reassembly during the cell cycle. Two processes occur during Golgi disassembly: unstacking of the Golgi cisternae mediated by phosphorylation of the Golgi stacking proteins by mitotic kinases; and vesiculation of the Golgi cisternae by COPI vesicle budding. Reassembly of the Golgi fragments into Golgi stacks involves membrane fusion and restacking. Fusion is mediated by two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each functions together with specific adaptor proteins. Restacking is mediated by protein phosphatase PP2A that dephosphorylates the Golgi stacking proteins. Ubiqitination occurs during Golgi disassembly and is required for the subsequent reassembly mediated by p97/p47.

Golgi biogenesis: how to form a new Golgi when a cell divides?
Here we focus on the role of monoubiquitination in regulating Golgi membrane dynamics in the cell cycle. Several converging lines of evidence have suggested that monoubiquitination plays an essential role in the regulation of Golgi membrane dynamics in the cell cycle. Monoubiquitination, as a regulatory signal, occurs during mitotic Golgi disassembly and is required for subsequent Golgi reassembly (Fig. 4). The AAA ATPase p97 and its adapter protein p47 are involved in membrane fusion during post-mitotic Golgi reassembly. The p97/p47 complex binds to monoubiquitin through the UBA domain of p47, and this interaction is required for p97-mediated Golgi membrane fusion. Proteasome activity is not involved in either Golgi disassembly or reassembly. We have identified a Golgi-localized ubiquitin E3 ligase HACE1 which is involved in mitotic Golgi disassembly and discovered the p97/p47 binding protein VCIP135 as a deubiquitinating enzyme whose activity is required for post-mitotic Golgi reassembly. HACE1 is a tumor suppressor whose expression is down regulated in a number of tumors. We have also recently identified the substrates on the Golgi membranes whose ubiquitination and deubiquitination are regulated by HACE1 and VCIP135 in the cell cycle. These data suggest that cycles of addition and removal of ubiquitin to and from substrates regulate Golgi membrane dynamics during cell division. We are currently determining the coordination and regulation of these machineries in the biogenesis of the Golgi apparatus in the mammalian cell cycle.

Fig 5. AD Golgi rescue

Fig. 5. The Golgi is fragment upon Aβ accumulation and is rescued by inhibiting cdk5 and by expressing GRASP65. A-B. Inhibition of cdk5 reverses Aβ-induced Golgi fragmentation in neurons. Primary hippocampal neurons were treated with Aβ42 in the absence (A) or presence (B) of a cdk5 inhibitor and stained for the Golgi in green, red is a neuronal marker. C-D. Expression a nonphosphorylatable GRASP65 mutant rescues the Golgi structure in a cell model of Alzheimer’s disease. Cells stably expressing APPswe/PS1∆E9 were transfected with GFP (C) or a nonphosphorylatable GRASP65 mutant tagged with GFP (D). Note the intact Golgi in green cells in D (indicated by asterisks). Scale bar, 20 µm. E. Working hypothesis. APP expression and processing cause Aβ accumulation (1), which induces Golgi fragmentation through modification of Golgi structural proteins (2), which in turn, increases Aβ production by enhancing amyloidogenic cleavage (3). This deleterious feedback loop would impair the integrity of the secretory pathway and compromise neuronal function. Rescue of Golgi structure and function therefore may reduce Aβ production and thus delay AD development (4).

Golgi defect in diseases: cause, effect, and correction?
Golgi defects and dysfunctions of protein trafficking have been observed in many diseases. We have started to apply our expertise on Golgi structure formation to determine the cause of Golgi defect and its effects on the development of the disease, such as in AD, asthma and cancer. For example, Golgi fragmentation occurs in neurons of patients with Alzheimer’s disease (AD), but the underlying molecular mechanism causing the defects and the subsequent effects on disease development remain unknown. We have examined the Golgi structure in APPswe/PS1∆E9 transgenic mouse and tissue culture models and found that accumulation of amyloid beta peptides (Aβ) leads to Golgi fragmentation. Further biochemistry and cell biology studies revealed that Golgi fragmentation in AD is caused by phosphorylation of Golgi structural proteins such as GRASP65, which is induced by Aβ-triggered cdk5 activation. Significantly, inhibiting cdk5 or expressing nonphosphorylatable GRASP65 mutants both rescued the Golgi structure and reduced Aβ secretion by elevating α-cleavage of the amyloid precursor protein (APP). Our study provides a molecular mechanism for Golgi fragmentation and its effects on APP trafficking and processing in AD, suggesting Golgi as a potential drug target for AD treatment. In collaboration with Drs. Henry Paulson and Geoffery Murphy at UM medical school, we are currently testing whether rescue of the Golgi structure and function in an AD mouse model delays the disease development.

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