A role for clathrin in reassembly of the Golgi apparatus

The Golgi apparatus is a highly dynamic organelle whose organization is maintained by a proteinaceous matrix, cytoskeletal components, and inositol phospholipids. In mammalian cells, disassembly of the organelle occurs reversibly at the onset of mitosis and irreversibly during apoptosis. Several pharmacological agents including nocodazole, brefeldin A (BFA), and primary alcohols (1-butanol) induce reversible fragmentation of the Golgi apparatus. To dissect the mechanism of Golgi reassembly, rat NRK and GH3 cells were treated with 1-butanol, BFA, or nocodazole. During washout of 1-butanol, clathrin, a ubiquitous coat protein implicated in vesicle traffic at the trans-Golgi network and plasma membrane, and abundant clathrin coated vesicles were recruited to the region of nascent Golgi cisternae. Knockdown of endogenous clathrin heavy chain showed that the Golgi apparatus failed to reform efficiently after BFA or 1-butanol removal. Instead, upon 1-butanol washout, it maintained a compact, tight morphology. Our results suggest that clathrin is required to reassemble fragmented Golgi elements. In addition, we show that after butanol treatment the Golgi apparatus reforms via an initial compact intermediate structure that is subsequently remodeled into the characteristic interphase lace-like morphology and that reassembly requires clathrin.     

Reconstitution of the Golgi reassembly process in semi-intact MDCK cells

The Golgi apparatus, which consists of stacks of cisternae during interphase, is fragmented or dispersed throughout the cytoplasm at the onset of mitosis. A sea sponge metabolite, ilimaquinone (IQ), causes Golgi membranes to vesiculate. And after its removal, the vesiculated membranes reassemble into stacks of cisternae in the perinuclear region. To study the mechanism of Golgi membrane dynamics during mitosis, we have reconstituted the reassembly process of IQ-induced vesiculated Golgi membranes in streptolysin O-permeabilized Mardin-Darby canine kidney (MDCK) cells. Monitoring the dynamics of Golgi membranes labeled with a green fluorescence protein (GFP)-tagged protein, we dissected the process into two elementary components: the reassembly of vesiculated Golgi membranes into punctate structures; and the subsequent reformation of these structures into stacks of cisternae near the nucleus. Using morphometric analysis, we studied the kinetics and biochemical requirements for the process, and revealed that an NEM-sensitive factor, cytoplasmic dynein, and GTP binding protein were involved in the Golgi reassembly.     

Mitotic inheritance of the Golgi complex and its role in cell division

The Golgi apparatus plays essential roles in the processing and sorting of proteins and lipids, but it can also act as a signalling hub and a microtubule‐nucleation centre. The Golgi complex (GC) of mammalian cells is composed of stacks connected by tubular bridges to form a continuous membranous system. In spite of this structural complexity, the GC is highly dynamic, and this feature becomes particularly evident during mitosis, when the GC undergoes a multi‐step disassembly process that allows its correct partitioning and inheritance by daughter cells. Strikingly, different steps of Golgi disassembly control mitotic entry and progression, indicating that cells actively monitor Golgi integrity during cell division. Here, we summarise the basic mechanisms and the molecular players that are involved in Golgi disassembly, focussing in particular on recent studies that have revealed the fundamental signalling pathways that connect Golgi inheritance to mitotic entry and progression.     

A mitotic form of the Golgi apparatus in HeLa cells

Galactosyltransferase, a marker for trans-Golgi cisternae in interphase cells, was localized in mitotic HeLa cells embedded in Lowicryl K4M by immunoelectron microscopy. Specific labeling was found only over multivesicular structures that we term Golgi clusters. Unlike Golgi stacks in interphase cells, these clusters lacked elongated cisternae and ordered stacking of their components but did comprise two distinct regions, one containing electron-lucent vesicles and the other, smaller, vesiculo-tubular structures. Labeling for galactosyltransferase was found predominantly over the latter region. Both structures were embedded in a dense matrix that excluded ribosomes and the cluster was often bounded by cisternae of the rough endoplasmic reticulum, sometimes on all sides. Clusters were present at all stages of mitosis examined, which included prometaphase, metaphase, and telophase. They were also identified in conventionally processed mitotic cells and shown to contain another trans-Golgi marker, thiamine pyrophosphatase. Serial sectioning showed that clusters were discrete and globular and multiple copies appeared to be dispersed in the cytoplasm. Their possible role in the division of the Golgi apparatus is discussed.     

Coalescence of Golgi fragments in microtubule-deprived living cells

The process of stack coalescence, an important mechanism of Golgi recovery from mitosis, was examined using novel experimental paradigms. In living cells with disrupted (by nocodazole) microtubules, galactosyl transferase-GFP-labelled Golgi fragments constantly appeared, grew, sometimes moved with a speed of 1-2 microns/min, coalesced or gradually diminished and disappeared. The rate of Golgi fragment turnover and coalescence was highly balanced to maintain a constant number of Golgi units per cell. Moreover some Golgi islands appear and some received new GalTase-GFP after photobleaching of cell cytoplasm. Short tubules extending from the rims of scattered Golgi fragments frequently formed bridges between ministacks, inducing their coalescence. The frequency of coalescence could also be inhibited by disruption of actin microfilaments. After the Golgi redistribution into endoplasmic reticulum induced by brefeldin A, either the growth of small Golgi fragments or their coalescence leads to compartmentalized stack formation without the participation of microtubules. These results demonstrate that this coalescence between isolated Golgi stacks is microtubule-independent and could thus be mediated by membranous tubules.     

Mitotic inheritance of the Golgi complex

In mammals, the Golgi complex is structured in the form of a continuous membranous system composed of stacks connected by tubular bridges, the “Golgi ribbon”. At the onset of mitosis, the Golgi complex undergoes a multi-step fragmentation process that is required for its correct partition into the dividing cells. Regulation of Golgi fragmentation and cell cycle progression appear to be precisely coordinated. Here, we review recent studies that are revealing the fundamental mechanisms, the molecular players and the biological significance of the mitotic inheritance of the Golgi complex in mammalian cells.     

Microtubule independent vesiculation of Golgi membranes and the reassembly of vesicles into Golgi stacks

We have recently shown that ilimaquinone (IQ) causes the breakdown of Golgi membranes into small vesicles (VGMs for vesiculated Golgi membranes) and inhibits vesicular protein transport between successive Golgi cisternae (Takizawa et al., 1993). While other intracellular organelles, intermediate filaments, and actin filaments are not affected, we have found that cytoplasmic microtubules are depolymerized by IQ treatment of NRK cells. We provide evidence that IQ breaks down Golgi membranes regardless of the state of cytoplasmic microtubules. This is evident from our findings that Golgi membranes break down with IQ treatment in the presence of taxol stabilized microtubules. Moreover, in cells where the microtubules are first depolymerized by microtubule disrupting agents which cause the Golgi stacks to separate from one another and scatter throughout the cytoplasm, treatment with IQ causes further breakdown of these Golgi stacks into VGMs. Thus, IQ breaks down Golgi membranes independently of its effect on cytoplasmic microtubules. Upon removal of IQ from NRK cells, both microtubules and Golgi membranes reassemble. The reassembly of Golgi membranes, however, takes place in two sequential steps: the first is a microtubule independent process in which the VGMs fuse together to form stacks of Golgi cisternae. This step is followed by a microtubule-dependent process by which the Golgi stacks are carried to their perinuclear location in the cell. In addition, we have found that IQ has no effect on the structural organization of Golgi membranes at 16 degrees C. However, VGMs generated by IQ are capable of fusing and assembling into stacks of Golgi cisternae at 16 degrees C. This is in contrast to the cells recovering from BFA treatment where, after removal of BFA at 16 degrees C, resident Golgi enzymes fail to exit the ER, a process presumed to require the formation of vesicles. We propose that at 16 degrees C there may be general inhibition in the process of vesicle formation, whereas the process of vesicle fusion is not affected.     

The multiple facets of the Golgi reassembly stacking proteins

The mammalian GRASPs (Golgi reassembly stacking proteins) GRASP65 and GRASP55 were first discovered more than a decade ago as factors involved in the stacking of Golgi cisternae. Since then, orthologues have been identified in many different organisms and GRASPs have been assigned new roles that may seem disconnected. In vitro, GRASPs have been shown to have the biochemical properties of Golgi stacking factors, but the jury is still out as to whether they act as such in vivo. In mammalian cells, GRASP65 and GRASP55 are required for formation of the Golgi ribbon, a structure which is fragmented in mitosis owing to the phosphorylation of a number of serine and threonine residues situated in its C-terminus. Golgi ribbon unlinking is in turn shown to be part of a mitotic checkpoint. GRASP65 also seems to be the key target of signalling events leading to re-orientation of the Golgi during cell migration and its breakdown during apoptosis. Interestingly, the Golgi ribbon is not a feature of lower eukaryotes, yet a GRASP homologue is present in the genome of Encephalitozoon cuniculi, suggesting they have other roles. GRASPs have no identified function in bulk anterograde protein transport along the secretory pathway, but some cargo-specific trafficking roles for GRASPs have been discovered. Furthermore, GRASP orthologues have recently been shown to mediate the unconventional secretion of the cytoplasmic proteins AcbA/Acb1, in both Dictyostelium discoideum and yeast, and the Golgi bypass of a number of transmembrane proteins during Drosophila development. In the present paper, we review the multiple roles of GRASPs.     

Kin recognition between medial Golgi enzymes in HeLa cells.

The medial Golgi enzymes, N-acetylglucosaminyltransferase I (NAGT I) and mannosidase II (Mann II), and the trans Golgi enzyme, beta-1,4-galactosyltransferase (GalT) were each retained in the endoplasmic reticulum (ER) by grafting on the cytoplasmic tail of the p33 invariant chain. Transient and stable expression of p33/NAGT I in HeLa cells caused relocation of endogenous Mann II to the ER and transient expression of p33/Mann II had a similar effect on endogenous NAGT I. Neither of these endogenous medial enzymes were affected by transient expression of p33/GalT. These data provide strong evidence for kin recognition between medial Golgi enzymes and suggest a role for them in the organization of the Golgi stack.     

Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

Osmium impregnation was used to determine the number of Golgi apparatus in both interphase and mitotic HeLa cells. The number was found to increase substantially during mitosis to the point where random partitioning alone would explain the nearly equal numbers found in each daughter cell.     

Mitotic disassembly of the mammalian Golgi apparatus

A cell-free system that mimics mitotic fragmentation of Golgi stacks has provided a working model for the disassembly process. Two distinct pathways, one COP-dependent and one COP-independent, act on Golgi stacks to give rise to two types of end products: transport vesicles and larger, more heterogeneous vesicles and tubules. We suggest that both mitotic end products result from enhanced fission of Golgi membranes under conditions where membrane fusion is generally inhibited.     

Mitotic division of the mammalian Golgi apparatus

Successful cell reproduction requires faithful duplication and proper segregation of cellular contents, including not only the genome but also intracellular organelles. Since the Golgi apparatus is an essential organelle of the secretory pathway, its accurate inheritance is therefore of importance to sustain cellular function. Regulation of Golgi division and its coordination with cell cycle progression involves a series of sequential events that are subjected to a precise spatiotemporal control. Here, we summarize the current knowledge about the underlying mechanisms, the molecular players and the biological relevance of this process, particularly in mammalian cells, and discuss the unsolved problems and future perspectives opened by the recent studies.     

VCIP135 acts as a deubiquitinating enzyme during p97-p47-mediated reassembly of mitotic Golgi fragments

The AAA-ATPase p97/Cdc48 functions in different cellular pathways using distinct sets of adapters and other cofactors. Together with its adaptor Ufd1-Npl4, it extracts ubiquitylated substrates from the membrane for subsequent delivery to the proteasome during ER-associated degradation. Together with its adaptor p47, on the other hand, it regulates several membrane fusion events, including reassembly of Golgi cisternae after mitosis. The finding of a ubiquitin-binding domain in p47 raises the question as to whether the ubiquitin-proteasome system is also involved in membrane fusion events. Here, we show that p97-p47-mediated reassembly of Golgi cisternae requires ubiquitin, but is not dependent on proteasome-mediated proteolysis. Instead, it requires the deubiquitinating activity of one of its cofactors, VCIP135, which reverses a ubiquitylation event that occurs during mitotic disassembly. Together, these data reveal a cycle of ubiquitylation and deubiquitination that regulates Golgi membrane dynamics during mitosis. Furthermore, they represent the first evidence for a proteasome-independent function of p97/Cdc48.     

Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N- acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans- Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.     

Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.     

Cog3p depletion blocks vesicle-mediated Golgi retrograde trafficking in HeLa cells

The conserved oligomeric Golgi (COG) complex is an evolutionarily conserved multi-subunit protein complex that regulates membrane trafficking in eukaryotic cells. In this work we used short interfering RNA strategy to achieve an efficient knockdown (KD) of Cog3p in HeLa cells. For the first time, we have demonstrated that Cog3p depletion is accompanied by reduction in Cog1, 2, and 4 protein levels and by accumulation of COG complex-dependent (CCD) vesicles carrying v-SNAREs GS15 and GS28 and cis-Golgi glycoprotein GPP130. Some of these CCD vesicles appeared to be vesicular coat complex I (COPI) coated. A prolonged block in CCD vesicles tethering is accompanied by extensive fragmentation of the Golgi ribbon. Fragmented Golgi membranes maintained their juxtanuclear localization, cisternal organization and are competent for the anterograde trafficking of vesicular stomatitis virus G protein to the plasma membrane. In a contrast, Cog3p KD resulted in inhibition of retrograde trafficking of the Shiga toxin. Furthermore, the mammalian COG complex physically interacts with GS28 and COPI and specifically binds to isolated CCD vesicles.     

Golgi reassembly after mitosis: the AAA family meets the ubiquitin family

The Golgi apparatus in animal cells breaks down at the onset of mitosis and is later rebuilt in the two daughter cells. Two AAA ATPases, NSF and p97/VCP, have been implicated in regulating membrane fusion steps that lead to regrowth of Golgi cisternae from mitotic fragments. NSF dissociates complexes of SNARE proteins, thereby reactivating them to mediate membrane fusion. However, NSF has a second function in regulating SNARE pairing together with the ubiquitin-like protein GATE-16. p97/VCP, on the other hand, is involved in a cycle of ubiquitination and deubiquitination of an unknown target that governs Golgi membrane dynamics. Here, these findings are reviewed and discussed in the context of the increasingly evident role of ubiquitin in membrane traffic processes.     

Biosynthesis and modification of Golgi mannosidase II in HeLa and 3T3 cells

The biosynthesis and post-translational modification of mannosidase II, an enzyme required in the maturation of asparagine-linked oligosaccharides in the Golgi complex, has been investigated. Antibody raised against this enzyme purified from rat liver Golgi membranes was used to immunoprecipitate mannosidase II from rat liver, 3T3 cells, or HeLa cells. Mannosidase II immunoprecipitated from rat liver Golgi membranes, when analyzed by polyacrylamide gel electrophoresis, migrated with an apparent molecular weight of approximately 124,000. In contrast, the enzyme purified from rat liver Golgi membranes was shown to contain both the 124,000-dalton component and a 110,000-dalton polypeptide believed to result from degradation of intact mannosidase II during purification. Mannosidase II from 3T3 and HeLa cells migrated on polyacrylamide gels with apparent molecular weights of approximately 124,000 and 134,000-136,000, respectively. When immunoprecipitated from radiolabeled cultures, mannosidase II from both cell types was similar in the following respects: (a) the initial synthesis product had an apparent molecular weight of approximately 124,000; (b) in cultures treated with tunicamycin the initial synthesis product had an apparent molecular weight of approximately 117,000; (c) endoglycosidase H digestion of the initial synthesis product gave an apparent molecular weight similar to the tunicamycin-induced polypeptide; (d) the mature enzyme was mostly (HeLa) or entirely (3T3) resistant to digestion by endoglycosidase H. Loss of [35S]methionine from intracellular mannosidase II occurred with a half-life of approximately 20 h; there was no appreciable accumulation of labeled immuno-reactive material in the medium. HeLa mannosidase II, but not the 3T3 enzyme, was additionally modified 1-3 h after synthesis, the initial synthesis product being converted to a doublet with an apparent molecular weight of approximately 134,000-136,000. Evidence is presented that this mobility shift may result from O-glycosylation. Mannosidase II from both cell types could be labeled with [32P]phosphate or [35S]sulfate. The latter is apparently attached to oligosaccharide as indicated by inhibition of labeling by tunicamycin; the former was shown with the HeLa enzyme to be present as serine phosphate moieties. In addition, [3H]palmitate could be incorporated into the enzyme in 3T3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)