Danming Tang

ERK regulates Golgi and centrosome orientation towards the leading edge through GRASP65

Directed cell migration requires the orientation of the Golgi and centrosome toward the leading edge. We show that stimulation of interphase cells with the mitogens epidermal growth factor or lysophosphatidic acid activates the extracellular signal–regulated kinase (ERK), which phosphorylates the Golgi structural protein GRASP65 at serine 277. Expression of a GRASP65 Ser277 to alanine mutant or a GRASP65 1–201 truncation mutant, neither of which can be phosphorylated by ERK, prevents Golgi orientation to the leading edge in a wound assay. We show that phosphorylation of GRASP65 with recombinant ERK leads to the loss of GRASP65 oligomerization and causes Golgi cisternal unstacking. Furthermore, preventing Golgi polarization by expressing mutated GRASP65 inhibits centrosome orientation, which is rescued upon disassembly of the Golgi structure by brefeldin A. We conclude that Golgi remodeling, mediated by phosphorylation of GRASP65 by ERK, is critical for the establishment of cell polarity in migrating cells.

Properties of the Thioredoxin Fold Superfamily Are Modulated by a Single Amino Acid Residue

The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.

Golgi Cisternal Unstacking Stimulates COPI Vesicle Budding and Protein Transport

The Golgi apparatus in mammalian cells is composed of flattened cisternae that are densely packed to form stacks. We have used the Golgi stacking protein GRASP65 as a tool to modify the stacking state of Golgi cisternae. We established an assay to measure protein transport to the cell surface in post-mitotic cells in which the Golgi was unstacked. Cells with an unstacked Golgi showed a higher transport rate compared to cells with stacked Golgi membranes. Vesicle budding from unstacked cisternae in vitro was significantly increased compared to stacked membranes. These results suggest that Golgi cisternal stacking can directly regulate vesicle formation and thus the rate of protein transport through the Golgi. The results further suggest that at the onset of mitosis, unstacking of cisternae allows extensive and rapid vesiculation of the Golgi in preparation for its subsequent partitioning.

The Role of GRASP65 in Golgi Cisternal Stacking and Cell Cycle Progression

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.

Molecular Mechanism of Mitotic Golgi Disassembly and Reassembly Revealed by a Defined Reconstitution Assay

In mammalian cells, flat Golgi cisternae closely arrange together to form stacks. During mitosis, the stacked structure undergoes a continuous fragmentation process. The generated mitotic Golgi fragments are distributed into the daughter cells, where they are reassembled into new Golgi stacks. In this study, an in vitro assay has been developed using purified proteins and Golgi membranes to reconstitute the Golgi disassembly and reassembly processes. This technique provides a useful tool to delineate the mechanisms underlying the morphological change. There are two processes during Golgi disassembly: unstacking and vesiculation. Unstacking is mediated by two mitotic kinases, cdc2 and plk, which phosphorylate the Golgi stacking protein GRASP65 and thus disrupt the oligomer of this protein. Vesiculation is mediated by the COPI budding machinery ARF1 and the coatomer complex. When treated with a combination of purified kinases, ARF1 and coatomer, the Golgi membranes were completely fragmented into vesicles. After mitosis, there are also two processes in Golgi reassembly: formation of single cisternae by membrane fusion, and restacking. Cisternal membrane fusion requires two AAA ATPases, p97 and NSF (N-ethylmaleimide-sensitive fusion protein), each of which functions together with specific adaptor proteins. Restacking of the newly formed Golgi cisternae requires dephosphorylation of Golgi stacking proteins by the protein phosphatase PP2A. This systematic study revealed the minimal machinery that controls the mitotic Golgi disassembly and reassembly processes.

Cell cycle regulation of Golgi membrane dynamics

The Golgi apparatus is a membranous organelle in the cell that plays essential roles in protein and lipid trafficking, sorting, processing, and modification. Its basic structure is a stack of closely aligned flattened cisternae. In mammalian cells, dozens of Golgi stacks are often laterally linked into a ribbon-like structure. Biogenesis of the Golgi during cell division occurs through a sophisticated disassembly and reassembly process that can be divided into three distinct but cooperative steps, including the deformation and reformation of the Golgi cisternae, stacks, and ribbon. Here, we review our current understanding of the protein machineries that control these three steps in the cycle of mammalian cell division: GRASP65 and GRASP55 in Golgi stack and ribbon formation; ubiquitin and AAA ATPases in postmitotic Golgi membrane fusion; and golgins and cytoskeleton in Golgi ribbon formation.

The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle.

Partitioning of the Golgi membrane into daughter cells during mammalian cell division occurs through a unique disassembly and reassembly process that is regulated by ubiquitination. However, the identity of the ubiquitin ligase is unknown. Here we show that the Homologous to the E6-AP Carboxyl Terminus (HECT) domain containing ubiquitin ligase HACE1 is targeted to the Golgi membrane through interactions with Rab proteins. The ubiquitin ligase activity of HACE1 in mitotic Golgi disassembly is required for subsequent postmitotic Golgi membrane fusion. Depletion of HACE1 using small interfering RNAs or expression of an inactive HACE1 mutant protein in cells impaired postmitotic Golgi membrane fusion. The identification of HACE1 as a Golgi-localized ubiquitin ligase provides evidence that ubiquitin has a critical role in Golgi biogenesis during the cell cycle.

Reconstitution of the cell cycle-regulated Golgi disassembly and reassembly in a cell-free system

The Golgi apparatus undergoes extensive disassembly during mitosis and reassembly in post-mitotic daughter cells. This process has been mimicked in vitro by treating Golgi membranes with mitotic and interphase cytosol. To determine the minimal machinery that controls the morphological change, we have developed a defined Golgi disassembly and reassembly assay that reconstitutes this process using purified proteins instead of cytosol. Treatment of Golgi membranes with mitotic kinases and COPI coat proteins efficiently disassembles the membranes into mitotic Golgi fragments, whereas further incubation with p97 or N-ethylmaleimide-sensitive factor (two AAA ATPases involved in membrane fusion) and their cofactors, in combination with protein phosphatase PP2A, leads to reassembly of the membranes into new Golgi stacks. The whole process takes 3–4 d and is applicable for identification and determination of novel cytosolic and membrane proteins that regulate Golgi membrane dynamics in the cell cycle.

Sequential phosphorylation of GRASP65 during mitotic Golgi disassembly

Summary GRASP65 phosphorylation during mitosis and dephosphorylation after mitosis are required for Golgi disassembly and reassembly during the cell cycle. At least eight phosphorylation sites on GRASP65 have been identified, but whether they are modified in a coordinated fashion during mitosis is so far unknown. In this study, we raised phospho-specific antibodies that recognize phosphorylated T220/T224, S277 and S376 residues of GRASP65, respectively. Biochemical analysis showed that cdc2 phosphorylates all three sites, while plk1 enhances the phosphorylation. Microscopic studies using these antibodies for double and triple labeling demonstrate sequential phosphorylation and dephosphorylation during the cell cycle. S277 and S376 are phosphorylated from late G2 phase through metaphase until telophase when the new Golgi is reassembled. T220/224 is not modified until prophase, but is highly modified from prometaphase to anaphase. In metaphase, phospho-T220/224 signal localizes on both Golgi haze and mitotic Golgi clusters that represent dispersed Golgi vesicles and Golgi remnants, respectively, while phospho-S277 and S376 labeling is more concentrated on mitotic Golgi clusters. Expression of a phosphorylation-resistant GRASP65 mutant T220A/T224A inhibited mitotic Golgi fragmentation to a much larger extent than the expression of the S277A and S376A mutants. In cytokinesis, T220/224 dephosphorylation occurs prior to that of S277, but after S376. This study provides evidence that GRASP65 is sequentially phosphorylated and dephosphorylated during mitosis at different sites to orchestrate Golgi disassembly and reassembly during cell division, with phosphorylation of the T220/224 site being most critical in the process.

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

GRASP65 plays a role in Golgi ribbon formation. Because the gaps between Golgi stacks are heterogeneous and large, it is possible that other proteins may help GRASP65 in ribbon linking. Mena is a novel GRASP65-binding protein that promotes actin elongation and enhances GRASP65 oligomerization to link Golgi stacks into a ribbon.