A Role for Clathrin in Reassembly of the Golgi Apparatus

Abstract

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. INTRODUCTIONThe Golgi apparatus is a polarized organelle that in interphase cells exists as a highly organized membranous network in the juxtanuclear region of the cell; it plays key roles in protein and lipid modification as well as cargo sorting. The structural organization of the Golgi apparatus is maintained by a family of high-molecular-weight proteins (e.g., GM130, GRASP65, Golgin-160, and p115), cytoskeletal components such as ␤III-spectrin as well as by the microtubule cytoskeleton (Nakamura et al., 1995;Barr et al., 1997;Stankewich et al., 1998;Shorter and Warren, 1999;Thyberg and Moskalewski, 1999). Several lipids, including phosphatidic acid (PA), diacylglycerol, phosphatidylinositol 4-phosphate [PtdIns(4)P], phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] have also been implicated in maintaining Golgi structure and function (De Matteis et al., 2005). In mammalian cells, the Golgi apparatus has a characteristic lace-like morphology that disassembles into vesicles and tubules at the onset of mitosis and reforms in telophase after the equal distribution of Golgi-derived vesicles into daughter cells. In contrast, during apoptosis the organelle fragments irreversibly (Machamer, 2003). Two models have been proposed to explain the inheritance of the Golgi complex. One suggests that free mitotic tubulovesicular clusters (MGCs) are the unit of Golgi inheritance (Jokitalo et al., 2001;Axelsson and Warren, 2004), whereas a second model proposes that the Golgi is inherited through a merged endoplasmic reticulum (ER)/Golgi compartment (Zaal et al., 1999). Furthermore, Seemann et al. (2002) suggested that the Golgi matrix provides a scaffold for reformation of the membranous ...

Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension

“…Loss of syntaxin-4 from the plasma membrane and its increased association in Golgi membranes would be expected to decrease effectiveness of Golgi-plasma membrane anterograde transport. Figure 3B also shows clathrin, an important mediator of export from the Golgi (46), is also trapped in MCTP-treated PAEC Golgi membranes. Figure 4 demonstrates that the trapping of Golgi tethers, SNAREs, and SNAPs is not cell type specific but also occurs in the A549 epithelial cells.…”

“…Additionally, because the process of membrane fusion is not dependent exclusively on SNAREs but also requires tether proteins, which are believed to act before SNARE interactions, we also investigated MCTP-induced changes in the Golgi tethers p115, GM130, giantin, and golgin 84, which play a critical role in association of coatomer protein complex I (COP-I) vesicles with the Golgi apparatus and ER-Golgi and intra-Golgi transport (4,51,52). Additionally, as transport of proteins from the trans-Golgi network is dependent on clathrin, we included clathrin in our investigations (46). Although defects in these critical mediators of vesicular trafficking through the Golgi apparatus have been described individually and have widespread effects on subcellular trafficking [as examples, mutant syntaxin-6 isoforms reduce the amount of cav-1 present at the plasma membrane in human skin fibroblasts (7), dominant negative GM130 inhibits COP-I vesicle fusion with the Golgi and decreases VSV-G cell surface trafficking (52), knockdown of GS15 largely leads to redistribution of GS28 and syntaxin-5, but not p115, GM130, and syntaxin-6 (70)], we observed that in MCTP-induced megalocytosis, all tethers, SNAREs, and SNAPs examined were trapped in the Golgi as assayed using cell fractionation and assumed a circumnuclear/perinuclear localization by immunofluorescence (Fig.…”

Clathrin Is Spindle-Associated but Not Essential for Mitosis

“…Clathrin has been implicated in a number of nonendocytic functions including the maintenance of basolateral polarity involving the regulation of protein exit from the Golgi [15], and post-mitotic Golgi reassembly [16]. A number of proteins required for the maintenance of the Golgi stack are known to associate with the spindle [17] and clathrin redistribution may be a marker for their recruitment.…”

The Golgi Protein p115 Associates with γ-Tubulin and Plays a Role in Golgi Structure and Mitosis Progression

“…Mitotic cells in cytokinesis were collected 10 h after release from the aphidicolin block, pelleted, and fixed as above. The cell pellets were processed for EM as described previously (37).…”