The Golgi complex is a membrane-bound organelle that serves as a central conduit for the processing of membrane and secretory proteins in all eukaryotic cells. The structure of the Golgi apparatus is seen in the stacks of flattened cisternae. However, the molecular machinery organizing the cisternae into stacks is poorly understood. Studying the biogenesis and molecular organization of this organelle is fundamental to understanding the mechanism of protein trafficking under normal conditions, and is critical to understanding many diseases caused by abnormal protein trafficking, processing and secretion. Research in our lab focuses on the molecular organization and biogenesis of the Golgi during the cell cycle. In particular, we are interested in studying the proteins that help "glue" the cisternae together in interphase cells and the role of ubiquitin in biogenesis of the Golgi apparatus in mammalian cells.
The system: the Golgi disassembly and reassembly assay
During each cycle of cell division, the Golgi apparatus must grow and divide into the two daughter cells. At the onset of mitosis, the Golgi apparatus undergoes continuous fragmentation and is broken down to vesicles, which are partitioned equally between the two daughter cells where they subsequently fuse to form a new Golgi apparatus. Using Golgi stacks purified from rat liver and cytosol prepared from HeLa cells, an in vitro Golgi disassembly/reassembly assay has been setup to reconstitute the breakdown and reassembly process of the Golgi during the cell cycle. This assay has been recently improved by replacement of the cytosol with purified proteins (Fig. 1). Mitotic Golgi disassembly can be efficiently mimicked using purified kinases and the COPI coat proteins. Two kinases, cdc2 and polo-like kinase (plk), unstack the cisternae via phosphorylation of the GRASP stacking proteins. ARF and coatomer vesiculate the cisternae by COPI vesicle budding. Golgi reassembly can be reconstituted using the two AAA ATPases, NSF and p97 (with their adapter proteins), p115, and protein phosphatase PP2A, which dephosphorylates GRASP proteins after mitosis. This improved system defines the minimal machinery for mitotic Golgi disassembly and reassembly, and can be used to identify and characterize novel proteins involved in Golgi membrane organization.
Fig. 1. EM photographs of Golgi disassembly and reassembly using purified components. Purified Golgi stacks were completely fragmented when incubated with purified kinases cdc2 and plk, with ARF and coatomer. New Golgi stacks could then be reassembled from these fragments when further treated with purified proteins in either the NSF pathway or the p97 pathway.
Golgi stacking proteins and the mechanism of stacking
GRASP65 was discovered as a Golgi stacking factor using the cell-free assay. Our recent work showed that GRASP65 forms homodimers, and that dimers from adjacent membranes oligomerize in a mitotically regulated manner. Trans-oligomerization of GRASP65 as such is both necessary and sufficient to hold the Golgi cisternae together. We have devised a bead assay to mimic cisternal stacking. In this assay, purified GRASP65 homodimers were coated onto the surface of Dynal magnetic beads. These beads aggregate extensively when treated with interphase cytosol (Fig. 2). The aggregates dissociated upon treatment with purified recombinant mitotic kinases, which could largely mimic mitotic cytosol for GRASP65 phosphorylation. Subsequent dephosphorylation of GRASP65 by interphase cytosol led to reaggregation. This cycle of dis-aggregation and re-aggregation mimics the mitotic unstacking (or fragmentation) and restacking (or reassembly) of Golgi membranes and provides the best evidence to date that GRASP65 can directly link adjacent surfaces. We propose that mitotically regulated trans-oligomerization of GRASP65 is the key mechanism for Golgi stacking (Fig. 2). GRASP65 is localized to cis cisternae, whereas GRASP55 is more medial-trans. We are currently characterizing GRASP55 to determine the relative contributions of the two GRASP proteins to Golgi architecture in mammalian cells. Our goal is to understand how this unique structure is organized at the molecular level and what the functional role of Golgi membrane stacking is.
Fig. 2. Beads assay for detecting trans-oligomers of GRASP65. Beads coated with recombinant GRASP65 aggregate when treated with bovine serum albumin (BSA) or with interphase cytosol (IC). Subsequent treatment with mitotic kinases (IC kinases) breaks up the aggregates, and subsequent dephosphorylation (IC kinases IC), mediated by phosphatases in IC, leads to reaggregation. Bar, 5 µm.
The role of p97 and ubiquitin in cell cycle regulation of
Golgi membrane dynamics
The complex of the AAA ATPase p97 (yeast cdc48) and its adapter protein p47 are involved in membrane fusion during Golgi reassembly. Binding of p47 to ubiquitin by a UBA domain is required for this process. Our recent results showed that ubiquitination occurs, as a regulatory signal, during disassembly and is required for subsequent reassembly. We have identified a Golgi localized ubiquitin E3 ligase that adds ubiquitin to an unidentified target during mitotic disassembly. This target is then recognized by p47 and the ubiquitin is removed by VCIP135. Proteasomes are not involved at any stage. We propose a cycle of ubiquitination and deubiquitination that regulates the mitotic Golgi cycle, perhaps by directing the p97 complex to those membranes that undergo fusion to form cisternae. We are further characterizing this regulatory mechanism and trying to identify the substrate(s) for this enzyme. Our aim is to understand the mechanism that fragments the Golgi early in mitosis and rebuilds it in each daughter cell towards the end of cell division.
Fig. 3. 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 fragmentation 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 of which functions together with specific adaptor proteins. Restacking is mediated by protein phosphatases that dephosphorylate the Golgi stacking proteins. Ubiqitination occurs during Golgi disassembly and is required for the subsequent reassembly mediated by p97/p47.
