Myristoylation Restricts Orientation of the GRASP Domain on Membranes and Promotes Membrane Tethering

The mammalian Golgi reassembly stacking protein (GRASP) proteins are Golgi-localized homotypic membrane tethers that organize Golgi stacks into a long, contiguous ribbon-like structure. It is unknown how GRASPs undergo trans pairing given that cis interactions between the proteins in the plane of the membrane are intrinsically favored. To test the hypothesis that myristoylation of the self-interacting GRASP domain restricts its orientation on the membrane to favor trans pairing, we established an in vitro assay that recapitulates GRASP-dependent membrane tethering and used neutron reflection under similar conditions to determine the orientation of the GRASP domain. In vivo, the membrane association of GRASP proteins is conferred by the simultaneous insertion of an N-terminal myristic acid and binding to a Golgi-associated binding partner. In our assay, the latter contact was replaced using a C-terminal hexa-His moiety, which bound to Ni²⁺-conjugated lipids incorporated into a substrate-supported bilayer lipid membrane. Nonmyristoylated protein lacked a fixed orientation on the membrane and inefficiently tethered liposomes. In contrast, myristoylated GRASP promoted tethering and exhibited a unique membrane complex. Thus, myristoylation restricts the membrane orientation of the GRASP domain favoring interactions in trans for membrane tethering.     

GRASP55 regulates intra‐Golgi localization of glycosylation enzymes to control glycosphingolipid biosynthesis

The Golgi apparatus, the main glycosylation station of the cell, consists of a stack of discontinuous cisternae. Glycosylation enzymes are usually concentrated in one or two specific cisternae along the cis‐trans axis of the organelle. How such compartmentalized localization of enzymes is achieved and how it contributes to glycosylation are not clear. Here, we show that the Golgi matrix protein GRASP55 directs the compartmentalized localization of key enzymes involved in glycosphingolipid (GSL) biosynthesis. GRASP55 binds to these enzymes and prevents their entry into COPI‐based retrograde transport vesicles, thus concentrating them in the trans‐Golgi. In genome‐edited cells lacking GRASP55, or in cells expressing mutant enzymes without GRASP55 binding sites, these enzymes relocate to the cis‐Golgi, which affects glycosphingolipid biosynthesis by changing flux across metabolic branch points. These findings reveal a mechanism by which a matrix protein regulates polarized localization of glycosylation enzymes in the Golgi and controls competition in glycan biosynthesis.     

Structural Insight into Golgi Membrane Stacking by GRASP65 and GRASP55 Proteins

The stacking of Golgi cisternae involves GRASP65 and GRASP55. The oligomerization of the N-terminal GRASP domain of these proteins, which consists of two tandem PDZ domains, is required to tether the Golgi membranes. However, the molecular basis for GRASP assembly is unclear. Here, we determined the crystal structures of the GRASP domain of GRASP65 and GRASP55. The structures reveal similar homotypic interactions: the GRASP domain forms a dimer in which the peptide-binding pockets of the two neighboring PDZ2 domains face each other, and the dimers are further connected by the C-terminal tail of one GRASP domain inserting into the binding pocket of the PDZ1 domain in another dimer. Biochemical analysis suggests that both types of contacts are relatively weak but are needed in combination for GRASP-mediated Golgi stacking. Our results unveil a novel mode of membrane tethering by GRASP proteins and provide insight into the mechanism of Golgi stacking.     

A novel imaging method for quantitative Golgi localization reveals differential intra-Golgi trafficking of secretory cargoes

Cellular functions of the Golgi are determined by the unique distribution of its resident proteins. Currently, electron microscopy is required for the localization of a Golgi protein at the sub-Golgi level. We developed a quantitative sub-Golgi localization method based on centers of fluorescence masses of nocodazole-induced Golgi ministacks under conventional optical microscopy. Our method is rapid, convenient, and quantitative, and it yields a practical localization resolution of ∼30 nm. The method was validated by the previous electron microscopy data. We quantitatively studied the intra-Golgi trafficking of synchronized secretory membrane cargoes and directly demonstrated the cisternal progression of cargoes from the cis- to the trans-Golgi. Our data suggest that the constitutive efflux of secretory cargoes could be restricted at the Golgi stack, and the entry of the trans-Golgi network in secretory pathway could be signal dependent.     

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.     

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.     

Structure of the membrane-tethering GRASP domain reveals a unique PDZ ligand interaction that mediates Golgi biogenesis

Biogenesis of the ribbon-like membrane network of the mammalian Golgi requires membrane tethering by the conserved GRASP domain in GRASP65 and GRASP55, yet the tethering mechanism is not fully understood. Here, we report the crystal structure of the GRASP55 GRASP domain, which revealed an unusual arrangement of two tandem PDZ folds that more closely resemble prokaryotic PDZ domains. Biochemical and functional data indicated that the interaction between the ligand-binding pocket of PDZ1 and an internal ligand on PDZ2 mediates the GRASP self-interaction, and structural analyses suggest that this occurs via a unique mode of internal PDZ ligand recognition. Our data uncover the structural basis for ligand specificity and provide insight into the mechanism of GRASP-dependent membrane tethering of analogous Golgi cisternae.     

Mena–GRASP65 interaction couples actin polymerization to Golgi ribbon linking

In mammalian cells, the Golgi reassembly stacking protein 65 (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers through the N-terminal GRASP domain. Because the GRASP domain is globular and relatively small, but the gaps between stacks are large and heterogeneous, it remains puzzling how GRASP65 physically links Golgi stacks into a ribbon. To explore the possibility that other proteins may help GRASP65 in ribbon linking, we used biochemical methods and identified the actin elongation factor Mena as a novel GRASP65-binding protein. Mena is recruited onto the Golgi membranes through interaction with GRASP65. Depleting Mena or disrupting actin polymerization resulted in Golgi fragmentation. In cells, Mena and actin were required for Golgi ribbon formation after nocodazole washout; in vitro, Mena and microfilaments enhanced GRASP65 oligomerization and Golgi membrane fusion. Thus Mena interacts with GRASP65 to promote local actin polymerization, which facilitates Golgi ribbon linking.     

Rab1 interaction with a GM130 effector complex regulates COPII vesicle cis--Golgi tethering

Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.     

The Golgi puppet master: COG complex at center stage of membrane trafficking interactions

The central organelle within the secretory pathway is the Golgi apparatus, a collection of flattened membranes organized into stacks. The cisternal maturation model of intra-Golgi transport depicts Golgi cisternae that mature from cis to medial to trans by receiving resident proteins, such as glycosylation enzymes via retrograde vesicle-mediated recycling. The conserved oligomeric Golgi (COG) complex, a multi-subunit tethering complex of the complexes associated with tethering containing helical rods family, organizes vesicle targeting during intra-Golgi retrograde transport. The COG complex, both physically and functionally, interacts with all classes of molecules maintaining intra-Golgi trafficking, namely SNAREs, SNARE-interacting proteins, Rabs, coiled-coil tethers, vesicular coats, and molecular motors. In this report, we will review the current state of the COG interactome and analyze possible scenarios for the molecular mechanism of the COG orchestrated vesicle targeting, which plays a central role in maintaining glycosylation homeostasis in all eukaryotic cells.     

A Model for the Self-Organization of Vesicular Flux and Protein Distributions in the Golgi Apparatus

The generation of two non-identical membrane compartments via exchange of vesicles is considered to require two types of vesicles specified by distinct cytosolic coats that selectively recruit cargo, and two membrane-bound SNARE pairs that specify fusion and differ in their affinities for each type of vesicles. The mammalian Golgi complex is composed of 6–8 non-identical cisternae that undergo gradual maturation and replacement yet features only two SNARE pairs. We present a model that explains how distinct composition of Golgi cisternae can be generated with two and even a single SNARE pair and one vesicle coat. A decay of active SNARE concentration in aging cisternae provides the seed for a cis trans SNARE gradient that generates the predominantly retrograde vesicle flux which further enhances the gradient. This flux in turn yields the observed inhomogeneous steady-state distribution of Golgi enzymes, which compete with each other and with the SNAREs for incorporation into transport vesicles. We show analytically that the steady state SNARE concentration decays exponentially with the cisterna number. Numerical solutions of rate equations reproduce the experimentally observed SNARE gradients, overlapping enzyme peaks in cis, medial and trans and the reported change in vesicle nature across the Golgi: Vesicles originating from younger cisternae mostly contain Golgi enzymes and SNAREs enriched in these cisternae and extensively recycle through the Endoplasmic Reticulum (ER), while the other subpopulation of vesicles contains Golgi proteins prevalent in older cisternae and hardly reaches the ER.     

GRASPing for consensus about the Golgi apparatus

Cisternae of the Golgi apparatus adhere to each other to form stacks, which are aligned side by side to form the Golgi ribbon. Two proteins, GRASP65 and GRASP55, previously implicated in stacking of cisternae, are shown to be required for the formation of the Golgi ribbon.

IntroductionThe Golgi apparatus is an intermediate organelle along the secretory pathway that receives proteins and lipids (“cargo”) from the endoplasmic reticulum, covalently modifies them, and then exports them via transport vesicles for trafficking to the plasma membrane or other organelles. In most eukaryotic cells, disc-shaped membrane cisternae, each containing a distinct repertoire of cargo-processing enzymes, are stacked one on top of another to form the “Golgi stack,” a visual hallmark of the organelle (Fig. 1). The cisternae of the Golgi stack are polarized, with the compartment receiving endoplasmic reticulum–derived cargo termed the cis cisterna followed by the medial; trans; and finally, the trans-Golgi network. The physiological advantages conferred by stacking of Golgi cisternae are unclear, but it is thought to enhance the efficiencies of the sequential chemical modifications of glycoproteins and glycolipids during secretion. Cultured mammalian cells may possess more than 100 Golgi stacks, which are aligned side by side about the centrosome to form the “Golgi ribbon” (Fig. 1). Vesicles and tubules span the intervening, “noncompact” zones between stacks of cisternae, connecting analogous cisternae across the ribbon and thereby ensuring a homogeneous distribution of Golgi resident proteins among all cisternae. During mitosis, the Golgi ribbon is unlinked, the stacks are disassembled, and the cisternae are converted to vesicles and tubules; after cytokinesis, the process is reversed, and the Golgi is rebuilt. The dynamic nature of Golgi structure in interphase and mitotic cells implies the existence of a reversible mechanism that tethers Golgi cisternae to each other to form the stack and a mechanism that aligns and links the stacks into the ribbon.Open in a separate windowFigure 1.The organization of the Golgi apparatus in vertebrate cells. Individual stacks of Golgi cisternae are aligned side to side to form the Golgi ribbon. The GRASP65 and GRASP55 proteins are depicted to be enriched on the rims of the indicated cisternae within individual stacks of cisternae, where they are required to maintain the arrangement of stacks into the ribbon.GRASP proteins tether Golgi cisternae in vitroInvestigations into the molecular basis of Golgi cisterna stacking have ultimately focused attention on a handful of cytoplasmic proteins called “Golgins” and “GRASPs” that are associated with specific Golgi cisternae and interact with each other. Of particular interest are two related proteins GRASP65 and GRASP55 (respective systematic names GORASP1 and GORASP2), discovered by Warren and colleagues via in vitro reconstitution experiments, as capable of mediating stacking of Golgi cisternae (Barr et al., 1997; Shorter et al., 1999). Whereas GRASP65 localizes to the cis cisterna, GRASP55 localization favors medial/trans Golgi cisternae (Shorter et al., 1999); hence, these proteins could, in principle, tether cisternae to form a minimal Golgi stack. In these in vitro assays, perturbations (mutations, antibody interference) to either GRASP65 or GRASP55 inhibited stacking of reformed Golgi cisternae. Moreover, GRASP proteins are phosphorylated in mitosis just before vesiculation of Golgi cisternae, and preventing phosphorylation impairs the disassembly of the Golgi apparatus and mitotic progression (Wang et al., 2003). These findings underpin models of the Golgi stack where GRASP65 and GRASP55, along with Golgin proteins, constitute the core components of a cytoplasmic “matrix” of proteins that surround the cisternae, mediating their stacking as well as the tethering of transport vesicles to cisternae. Curiously, plant cells contain stacked Golgi cisternae, yet they do not express any GRASP or GRASP-related proteins. And some nonvertebrate organisms with stacked Golgi cisternae express just one GRASP-related protein, while the Golgi cisternae are not stacked in other nonvertebrate organisms (e.g., yeast) that express a single GRASP (Glick and Malhotra, 1998). Apparently, the presence or number of GRASP proteins expressed does not correlate with stacked cisternae.Whereas the results of in vitro biochemical assays underpin our conceptions of GRASP protein function, probing their roles in vivo has proven to be quite challenging. First, depletion/deletion of each individual GRASP protein is largely without effect on Golgi stack or ribbon formation, but a very complex phenotype results from depletion/deletion of both GRASP proteins. Thus, some reports conclude that the GRASP proteins function redundantly to stack cisternae (Bekier et al., 2017), while others conclude that the Golgi ribbon, not the stack per se, is perturbed upon loss of GRASP proteins (Puthenveedu et al., 2006; Feinstein and Linstedt, 2008; Xiang and Wang, 2010; Lee et al., 2014; Veenendaal et al., 2014). Recently, two papers published in the Journal of Cell Biology employed different methodologies to perturb GRASP protein functions in vivo (Grond et al., 2020; Zhang and Seemann, 2021), providing the most conclusive insight to date into the roles of GRASP proteins in Golgi structure.The Golgi ribbon is unlinked upon loss of GRASP proteinsRabouille and colleagues used traditional mouse gene knockout technology to delete GRASP65, finding that such mice are viable with no apparent physiological deficits or gross morphological perturbations of the Golgi (Veenendaal et al., 2014). In their recent study (Grond et al., 2020), GRASP55 was deleted in the GRASP65 null background, but double-knockout mice could not be obtained, consistent with GRASP proteins being at least partially physiologically redundant. Next, using a conditional knockout approach, double GRASP null cells were produced postnatally in the small intestine, and the Golgi of intestinal epithelial cells was examined. In these cells, stacked Golgi cisternae were observed, but their arrangement into a ribbon was compromised, a result corroborated by more detailed analysis of cells in organoid cultures. These findings are at odds with the conclusions of Wang and colleagues (Bekier et al., 2017), who used CRISPR-Cas9 gene editing technology to construct cultured mammalian cell lines that do not express GRASP65 and GRASP55. They found that the appearance of Golgi cisternae was grossly altered, resembling clusters of tubules and vesicles (“tubulovesicular clusters”) about swollen cisterna remnants that debatably appeared to be stacked. One possible reason for the disparities between these two studies is that Bekier et al. (2017) documented that loss of GRASP proteins in cultured mammalian cells also resulted in depletion of a subset of Golgin proteins (e.g., GM130, Golgin-45) from Golgi cisternae, so it was not possible to parse the specific contributions of GRASP proteins to Golgi structure.Analyses of siRNA-depleted and gene-edited cell lines and modified animals are often complicated by incomplete depletion of a query protein, unintended loss of other proteins, or compensatory processes that obscure loss-of-function effects. Notably, siRNA depletion of GM130, which is associated with GRASP65 on the cis cisterna, impairs secretory traffic from the endoplasmic reticulum to the Golgi apparatus, resulting in a reduction in the size of Golgi cisternae and diminished interstack connectivity possibly due to vesiculation of cisternae (Seemann et al., 2000; Puthenveedu et al., 2006). To minimize these drawbacks, Zhang and Seemann (2021) used gene editing to modify the GRASP65 and GRASP55 loci to append an inducible protein degradation domain to each protein in cultured mammalian cells, which was used to elicit degradation of the GRASP proteins within just 2 h. Hence, the acute effects of GRASP protein depletion could be determined before the onset of potentially confounding effects. Fluorescence recovery after photobleaching assays of a fluorescently tagged Golgi resident protein revealed that acute depletion of both GRASP65 and GRASP55 resulted in decreased mobility of the resident Golgi enzyme within the ribbon, indicating that connectivity of cisternae between stacks was compromised. Stacks of Golgi cisternae with proper cis–trans polarity were observed by electron and light microscopy, both shortly (∼2 h) after GRASP protein turnover was initiated, and after mitosis, indicating that GRASP proteins are not required to establish or to maintain the Golgi stacks. Importantly, the authors observed no changes in the levels of GRASP-associated proteins (e.g., GM130) when assayed shortly after initiating GRASP protein turnover, but the amounts of several GRASP-associated proteins were reduced after prolonged growth in the absence of GRASP proteins. The results are in general agreement with experiments by Jarvela and Linstedt (2014), who expressed GRASP65 and GRASP55 fusion proteins appended with “killer RFP” and used chomophore-assisted light inactivation to rapidly (1 min) ablate the proteins in cultured mammalian cells. Similar to Zhang and Seemann (2021), they observed that the Golgi ribbon was disassembled upon inactivation of GRASP proteins, but stacking of cisternae was unaffected. Taken together, these results conclusively show that acute depletion of GRASP65 and GRASP55 impairs lateral linking of stacked Golgi cisternae within the ribbon while not affecting stacking of cisternae.Conclusions and perspectivesA body of work now more than 20 years old has shown that GRASP65 and GRAPS55 are core structural components of a matrix of cytoplasmic proteins associated with Golgi cisternae; however, the Grond et al. (2020) and Zhang and Seemann (2021) reports now firmly establish that GRASP proteins are dispensable for stacking of Golgi cisterna and indicate that they are required for linking Golgi stacks within the ribbon. These new studies suggest that the integrity of the Golgi matrix critically depends on the presence of GRASP proteins, and their absence perturbs the balance of cargo flow through the Golgi, reducing the interstack exchange required to maintain connectivity of stacks within the ribbon. How might GRASP proteins facilitate linking of stacks within the Golgi ribbon? When the ribbon is disrupted (using the microtubule depolymerizing reagent nocodazole) and individual Golgi stacks are examined, GRASP65 and GRASP55 appear to be enriched at the rims of Golgi cisternae (Fig. 1; Tie et al., 2018). Hence, the GRASP proteins are positioned at the vesicle-rich interface between adjacent cisternal stacks. Grond et al. (2020) observed reductions in the size of Golgi cisternae in cells deleted of both GRASP proteins and speculated that this may be due to increased coatomer I vesicle formation at the rims of cisternae. In this view, GRASP proteins dampen vesicle flux at the rims of Golgi cisternae, a model supported by the observation that depletion of GRASP proteins leads to an increase in secretion rate (Wang et al., 2008). These new studies firmly shift our view of GRASP protein function away from the stacking of Golgi cisternae, and we look forward to new mechanistic insights into the roles of GRASP proteins in Golgi ribbon formation as well as in non–Golgi-dependent processes, such as unconventional protein secretion (Kinseth et al., 2007).     

Functional compartmentation of the Golgi apparatus of plant cells : immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells

The Golgi apparatus of plant cells is engaged in both the processing of glycoproteins and the synthesis of complex polysaccharides. To investigate the compartmentalization of these functions within individual Golgi stacks, we have analyzed the ultrastructure and the immunolabeling patterns of high-pressure frozen and freeze-substituted suspension-cultured sycamore maple (Acer pseudoplatanus L.) cells. As a result of the improved structural preservation, three morphological types of Golgi cisternae, designated cis, medial, and trans, as well as the trans Golgi network, could be identified. The number of cis cisternae per Golgi stack was found to be fairly constant at approximately 1, whereas the number of medial and trans cisternae per stack was variable and accounted for the varying number of cisternae (3-10) among the many Golgi stacks examined. By using a battery of seven antibodies whose specific sugar epitopes on secreted polysaccharides and glycoproteins are known, we have been able to determine in which types of cisternae specific sugars are added to N-linked glycans, and to xyloglucan and polygalacturonic acid/rhamnogalacturonan-I, two complex polysaccharides. The findings are as follows. The β-1,4-linked d-glucosyl backbone of xyloglucan is synthesized in trans cisternae, and the terminal fucosyl residues on the trisaccharide side chains of xyloglucan are partly added in the trans cisternae, and partly in the trans Golgi network. In contrast, the polygalacturonic/rhamnogalacturonan-I backbone is assembled in cis and medial cisternae, methylesterification of the carboxyl groups of the galacturonic acid residues in the polygalacturonic acid domains occurs mostly in medial cisternae, and arabinose-containing side chains of the polygalacturonic acid domains are added to the nascent polygalacturonic acid/rhamnogalacturonan-I molecules in the trans cisternae. Double labeling experiments demonstrate that xyloglucan and polygalacturonic acid/rhamnogalacturonan-I can be synthesized concomitantly within the same Golgi stack. Finally, we show that the xylosyl residue-linked β-1,2 to the β-linked mannose of the core of N-linked glycans is added in medial cisternae. Taken together, our results indicate that in sycamore maple suspension-cultured cells, different types of Golgi cisternae contain different sets of glycosyl transferases, that the functional organization of the biosynthetic pathways of complex polysaccharides is consistent with these molecules being processed in a cis to trans direction like the N-linked glycans, and that the complex polysaccharide xyloglucan is assembled exclusively in trans Golgi cisternae and the trans Golgi network.     

Rapid degradation of GRASP55 and GRASP65 reveals their immediate impact on the Golgi structure

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.     

A GRASP55-rab2 effector complex linking Golgi structure to membrane traffic.

Membrane traffic between the endoplasmic reticulum (ER) and Golgi apparatus and through the Golgi apparatus is a highly regulated process controlled by members of the rab GTPase family. The GTP form of rab1 regulates ER to Golgi transport by interaction with the vesicle tethering factor p115 and the cis-Golgi matrix protein GM130, also part of a complex with GRASP65 important for the organization of cis-Golgi cisternae. Here, we find that a novel coiled-coil protein golgin-45 interacts with the medial-Golgi matrix protein GRASP55 and the GTP form of rab2 but not other Golgi rab proteins. Depletion of golgin-45 disrupts the Golgi apparatus and causes a block in secretory protein transport. These results demonstrate that GRASP55 and golgin-45 form a rab2 effector complex on medial-Golgi essential for normal protein transport and Golgi structure.     

Actin- and microtubule-dependent regulation of Golgi morphology by FHDC1

The Golgi apparatus is the central hub of intracellular trafficking and consists of tethered stacks of cis, medial, and trans cisternae. In mammalian cells, these cisternae are stitched together as a perinuclear Golgi ribbon, which is required for the establishment of cell polarity and normal subcellular organization. We previously identified FHDC1 (also known as INF1) as a unique microtubule-binding member of the formin family of cytoskeletal-remodeling proteins. We show here that endogenous FHDC1 regulates Golgi ribbon formation and has an apparent preferential association with the Golgi-derived microtubule network. Knockdown of FHDC1 expression results in defective Golgi assembly and suggests a role for FHDC1 in maintenance of the Golgi-derived microtubule network. Similarly, overexpression of FHDC1 induces dispersion of the Golgi ribbon into functional ministacks. This effect is independent of centrosome-derived microtubules and instead likely requires the interaction between the FHDC1 microtubule-binding domain and the Golgi-derived microtubule network. These effects also depend on the interaction between the FHDC1 FH2 domain and the actin cytoskeleton. Thus our results suggest that the coordination of actin and microtubule dynamics by FHDC1 is required for normal Golgi ribbon formation.     

A direct role for GRASP65 as a mitotically regulated Golgi stacking factor

Cell-free assays that mimic the disassembly and reassembly cycle of the Golgi apparatus during mitosis implicated GRASP65 as a mitotically regulated stacking factor. We now present evidence that GRASP65 is directly involved in stacking Golgi cisternae. GRASP65 is the major phosphorylation target in rat liver Golgi membranes of two mitotic kinases, cdc2-cyclin B and polo-like kinases, which alone will unstack Golgi membranes, generating single cisternae. Mitotic cells microinjected with antibodies to GRASP65 fail to form proper Golgi stacks after cell division. Beads coated with GRASP65 homodimers form extensive aggregates consistent with the formation of trans oligomers. These can be disaggregated using purified cdc2-cyclin B1 and polo-like kinases, and re-aggregated after dephosphorylation of GRASP65. Together, these data demonstrate that GRASP65 has the properties required to bind surfaces together in a mitotically regulated manner.     

Yeast Dop1 is required for glycosyltransferase retrieval from the trans-Golgi network

BackgroundGlycosyltransferases are type II membrane proteins that are responsible for glycan modification of proteins and lipids, and localize to distinct cisternae in the Golgi apparatus. During cisternal maturation, retrograde trafficking helps maintain the steady-state localization of these enzymes in the sub-compartments of the Golgi.MethodsTo understand how glycosyltransferases are recycled in the late Golgi complex, we searched for genes that are essential for budding yeast cell growth and that encode proteins localized in endosomes and in the Golgi. We specifically analyzed the roles of Dop1 and its binding partner Neo1 in retaining Golgi-resident glycosyltransferases, in the late Golgi complex.ResultsDop1 primarily localized to younger compartments of the trans-Golgi network (TGN) and seemed to cycle within the TGN. In contrast, Neo1, a P4-ATPase that interacts with Dop1, localized to the TGN. Abolition of DOP1 expression led to defects in the FM4-64 endocytic pathway. Dop1 and Neo1 were required for correct glycosylation of invertase, a secretory protein, at the Golgi. In DOP1-shutdown cells, Och1, a mannosyltransferase that is typically located in the cis-Golgi, mislocalized to the TGN. In addition, the function of multiple glycosyltransferases required for N- and O-glycosylation were impaired in DOP1-shutdown cells.ConclusionsOur results indicate that Dop1 is involved in vesicular transport at the TGN, and is critical for retrieving glycosyltransferases from the TGN to the Golgi in yeast.General significanceGolgi-resident glycosyltransferases recycling from the TGN to the Golgi is dependent on Dop1 and the P4-ATPase Neo1.     

The Pathway of Golgi Cluster Formation in Okadaic Acid-Treated Cells

Using stereology and immunoelectron microscopy we examined the pathway of Golgi duster formation during treatment with the phosphatase inhibitor okadaic acid. During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesicles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall reduction in membrane the total amount of immunolabeling for galactosyltransferase over the Golgi clusters of a typical cell was maintained, indicating that galactosyltransferase had been retained in Golgi membranes. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Golgi cisternae increased 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cisternae was selective. Careful evaluation of immunolabeled clusters showed that most of the galactosyltransferase labeling was located over complex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both connected to disassembling cisternae and within forming Golgi clusters, indicating that they were intermediates in cluster formation. We also investigated the role of vesicular transport in cluster formation. During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane depletion and stack disassembly. Taken together, our results indicate that during Golgi cluster formation, membrane leaves the Golgi but galactosyltransferase is retained within a tubular reticulum which is a direct descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus.     

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.