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.     

The dynamics of engineered resident proteins in the mammalian Golgi complex relies on cisternal maturation

After leaving the endoplasmic reticulum, secretory proteins traverse several membranous transport compartments before reaching their destinations. How they move through the Golgi complex, a major secretory station composed of stacks of membranous cisternae, is a central yet unsettled issue in membrane biology. Two classes of mechanisms have been proposed. One is based on cargo-laden carriers hopping across stable cisternae and the other on “maturing” cisternae that carry cargo forward while progressing through the stack. A key difference between the two concerns the behavior of Golgi-resident proteins. Under stable cisternae models, Golgi residents remain in the same cisterna, whereas, according to cisternal maturation, Golgi residents recycle from distal to proximal cisternae via retrograde carriers in synchrony with cisternal progression. Here, we have engineered Golgi-resident constructs that can be polymerized at will to prevent their recycling via Golgi carriers. Maturation models predict the progress of such polymerized residents through the stack along with cargo, but stable cisternae models do not. The results support the cisternal maturation mechanism.     

Evidence for an ATP-Dependent Proton Pump on the Golgi of Corn Coleoptiles

Corn (Zea mays L. cv Trojan T929) coleoptile membranes were fractionated on sucrose density gradients, and ATP-dependent proton pumping activity was localized by the techniques of [¹⁴C]methylamine uptake and quinacrine fluorescence quenching. Two peaks of proton pumping activity were detected: a light peak (1.07 grams/cubic centimeter) corresponding to the previously characterized tonoplast-type H⁺-ATPase, and a second peak (1.13 grams/cubic centimeter) which coincided with the Golgi markers, latent UDPase, and glucan synthase I. The second peak was lighter than that of the plasma membrane marker, uridine diphosphoglucose-sterol glucosyltransferase (1.16 grams/cubic centimeter) and was not inhibited by vanadate, an inhibitor of the plasma membrane ATPase. The activity was also better correlated with the Golgi cisternae marker, glucan synthase I, than with latent UDPase, a secretory vesicle marker, but a secretory vesicle location cannot be ruled out. The tonoplast-type and Golgi proton pumps were similar in several respects, including a pH optimum at 7.2, stimulation by chloride, inhibition by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide (DCCD), insensitivity to oligomycin and azide, and nucleotide specificity for Mg²⁺-ATP. However, the Golgi H⁺ pump was much less sensitive to nitrate and iodide, and more sensitive to the anion channel blockers, 4-acetamido-4′-isothiocyano-2,2′-stilbene sulfonic acid (SITS) and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (DIDS) than the tonoplast-type H⁺-pump. The Golgi pump, but not the tonoplast-type pump, was stimulated by valinomycin in the presence of KCl. It is concluded that the Golgi of corn coleoptiles contains a KCl-stimulated H⁺-ATPase which can acidify the interior of Golgi cisternae and associated vesicles.     

Intracellular Ca2+ stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2+ binding protein, calsequestrin, of Ca2+ ATPase and of the ER lumenal protein, Bip

To identify intracellular Ca2+ stores, we have mapped (by cryosection immunofluorescence and immunogold labeling) the distribution in the chicken cerebellar cortex of an essential component, the main low affinity-high capacity Ca2+ binding protein which in this tissue has been recently shown undistinguishable from muscle calsequestrin (Volpe, P., B. H. Alderson-Lang, L. Madeddu, E. Damiani, J. H. Collins, and A. Margreth. 1990. Neuron. 5:713-721). Appreciable levels of the protein were found exclusively within Purkinje neurons, distributed to the cell body, the axon, and the elaborate dendritic tree, with little labeling, however, of dendritic spines. At the EM level the protein displayed a dual localization: within the ER (rough- and smooth-surfaced cisternae, including the cisternal stacks recently shown [in the rat] to be highly enriched in receptors for inositol 1,4,5-triphosphate) and, over 10-fold more concentrated, within a population of moderately dense, membrane-bound small vacuoles and tubules, identified as calciosomes. These latter structures were widely distributed both in the cell body (approximately 1% of the cross-sectional area, particularly concentrated near the Golgi complex) and in the dendrites, up to the entrance of the spines. The distribution of calsequestrin was compared to those of another putative component of the Ca2+ stores, the membrane pump Ca2+ ATPase, and of the ER resident lumenal protein, Bip. Ca2+ ATPase was expressed by both calciosomes and regular ER cisternae, but excluded from cisternal stacks; Bip was abundant within the ER lumena (cisternae and stacks) and very low within calciosomes (average calsequestrin/Bip immunolabeling ratios were approximately 0.5 and 36.5 in the two types of structure, respectively). These results suggest that ER cisternal stacks do not represent independent Ca2+ stores, but operate coordinately with the adjacent, lumenally continuous ER cisternae. The ER and calciosomes could serve as rapidly exchanging Ca2+ stores, characterized however by different properties, in particular, by the greater Ca2+ accumulation potential of calciosomes. Hypotheses of calciosome biogenesis (directly from the ER or via the Golgi complex) are discussed.     

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat

Summary The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 M, was studied on the Golgi apparatus of absorptive cells in the small intestine of the rat. At 2–3 min after treatment most of the Golgi stacks exhibited dilated cisternae. At 4–5 min stacked cisternae were absent; they were replaced by groups of smooth-surfaced vacuoles. Dilatation and vacuolization occurred in the entire stacks without preferential effect on any particular Golgi subcompartment.Monensin did not influence the cytochemical Golgi reaction of thiamine pyrophosphatase and acid phosphatase. The characteristic staining pattern of these two enzymes in all Golgi cisternae of absorptive cells in the proximal small intestine, and the reactivity restricted to trans cisternae in distal segments of the small intestine, were unchanged after treatment with monensin. In the distal small intestine, the cytochemical pattern allowed the monensin-induced vacuoles to be attributed to the former cisor trans-Golgi face. Further, the cytochemical results demonstrate that vacuolization is not restricted to the stacked cisternae, but includes the trans-most cisterna. The latter, usually located at some distance from the Golgi stacks, has been defined as belonging to the GERL system in several types of cells. The clear response to monensin, an agent that selectively affects the Golgi apparatus, indicates common properties between trans-most and stacked Golgi cisternae.     

Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5–dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5–dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.     

Recycling of Golgi-resident Glycosyltransferases through the ER Reveals a Novel Pathway and Provides an Explanation for Nocodazole-induced Golgi Scattering

During microtubule depolymerization, the central, juxtanuclear Golgi apparatus scatters to multiple peripheral sites. We have tested here whether such scattering is due to a fragmentation process and subsequent outward tracking of Golgi units or if peripheral Golgi elements reform through a novel recycling pathway. To mark the Golgi in HeLa cells, we stably expressed the Golgi stack enzyme N-acetylgalactosaminyltransferase-2 (GalNAc-T2) fused to the green fluorescent protein (GFP) or to an 11–amino acid epitope, VSV-G (VSV), and the trans/TGN enzyme β1,4-galactosyltransferase (GalT) fused to GFP. After nocodazole addition, time-lapse microscopy of GalNAc-T2–GFP and GalT–GFP revealed that scattered Golgi elements appeared abruptly and that no Golgi fragments tracked outward from the compact, juxtanuclear Golgi complex. Once formed, the scattered structures were relatively stable in fluorescence intensity for tens of minutes. During the entire process of dispersal, immunogold labeling for GalNAc-T2–VSV and GalT showed that these were continuously concentrated over stacked Golgi cisternae and tubulovesicular Golgi structures similar to untreated cells, suggesting that polarized Golgi stacks reform rapidly at scattered sites. In fluorescence recovery after photobleaching over a narrow (FRAP) or wide area (FRAP-W) experiments, peripheral Golgi stacks continuously exchanged resident proteins with each other through what appeared to be an ER intermediate. That Golgi enzymes cycle through the ER was confirmed by microinjecting the dominant-negative mutant of Sar1 (Sar1p^dn) blocking ER export. Sar1p^dn was either microinjected into untreated or nocodazole-treated cells in the presence of protein synthesis inhibitors. In both cases, this caused a gradual accumulation of GalNAc-T2–VSV in the ER. Few to no peripheral Golgi elements were seen in the nocodazole-treated cells microinjected with Sar1p^dn. In conclusion, we have shown that Golgi-resident glycosylation enzymes recycle through the ER and that this novel pathway is the likely explanation for the nocodazole-induced Golgi scattering observed in interphase cells.     

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth

The proton-pumping ATPase (H⁺-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H⁺-ATPases, PMA2, an H⁺-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H⁺-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H⁺-ATPase with PMA4, an H⁺-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H⁺-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.     

Metabolism of Uridine 5′-Diphosphate-Glucose in Golgi Vesicles from Pea Stems

Uridine 5′-diphosphate-glucose (UDP-Glc) is transported into the lumen of the Golgi cisternae, where is used for polysaccharide biosynthesis. When Golgi vesicles were incubated with UDP-[³H]Glc, [³H]Glc was rapidly transferred to endogenous acceptors and UDP-Glc was undetectable in Golgi vesicles. This result indicated that a uridine-containing nucleotide was rapidly formed in the Golgi vesicles. Since little is known about the fate of the nucleotide derived from UDP-Glc, we analyzed the metabolism of the nucleotide moiety of UDP-Glc by incubating Golgi vesicles with [α-³²P]UDP-Glc, [β-³²P]UDP-Glc, and [³H]UDP-Glc and identifying the resulting products. After incubation of Golgi vesicles with these radiolabeled substrates we could detect only uridine 5′-monophosphate (UMP) and inorganic phosphate (Pi). UDP could not be detected, suggesting a rapid hydrolysis of UDP by the Golgi UDPase. The by-products of UDP hydrolysis, UMP and Pi, did not accumulate in the lumen, indicating that they were able to exit the Golgi lumen. The exit of UMP was stimulated by UDP-Glc, suggesting the presence of a putative UDP-Glc/UMP antiporter in the Golgi membrane. However, the exit of Pi was not stimulated by UDP-Glc, suggesting that the exit of Pi occurs via an independent membrane transporter.     

The tubular network of the Golgi apparatus

 Golgi apparatus of both plant and animal cells are characterized by an extensive system of approximately 30 nm diameter peripheral tubules. The total surface area of the tubules and associated fenestrae is thought to be approximately equivalent to that of the flattened portions of cisternae. The tubules may extend for considerable distances from the stacks. The tubules are continuous with the peripheral edges of the stacked cisternae, but the way they interconnect differs across the stack. In plant cells, for example, tubules associated with the near-cis and mid cisternae often begin to anastomose close to the peripheral edges of the stacked cisternae, whereas the tubules of the trans cisternae are less likely to anastomose and are more likely to be directly continuous with the peripheral edges of the stacked cisternae. Additionally, the tubules may blend gradually into fenestrae that surround some of the stack cisternae. Because of the large surface area occupied by tubules and fenestrae, it is reasonable to suppose that these components of the Golgi apparatus play a significant role in Golgi apparatus function. Tubules clearly interconnect closely adjacent stacks of the Golgi apparatus and may represent a communication channel to synchronize stack function within the cell. A feasible hypothesis is that tubules may be a potentially static component of the Golgi apparatus in contrast to the stacked cisternal plates which may turn over continuously. The coated buds associated with tubules may represent the means whereby adjacent Golgi apparatus stacks exchange carbohydrate-processing enzymes or where resident Golgi apparatus proteins are introduced into and out of the stack during membrane flow differentiation. The limited gradation of tubules from cis to medial to trans offers additional possibilities for functional specialization of Golgi apparatus in keeping with the hypothesis that tubules are repositories of resident Golgi apparatus proteins protected from turnover during the flow differentiation of the flattened saccules of the Golgi apparatus stack. Accepted: 3 November 1997     

Isolation and Characterization of Toluene-Sensitive Mutants from the Toluene-Resistant Bacterium Pseudomonas putida GM73

To understand the mechanism underlying toluene resistance of a toluene-tolerant bacterium, Pseudomonas putida GM73, we carried out Tn5 mutagenesis and isolated eight toluene-sensitive mutants. None of the mutants grew in the presence of 20% (vol/vol) toluene in growth medium but exhibited differential sensitivity to toluene. When wild-type cells were treated with toluene (1% [vol/vol]) for 5 min, about 2% of the cells could form colonies. In the mutants Ttg1, Ttg2, Ttg3, and Ttg8, the same treatment killed more than 99.9999% of cells (survival rate, <10⁻⁶). In Ttg4, Ttg5, Ttg6, and Ttg7, about 0.02% of cells formed colonies. We cloned the Tn5-inserted genes, and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by ttg genes were identified as follows. Ttg1 and Ttg2 are ATP binding cassette (ABC) transporter homologs; Ttg3 is a periplasmic linker protein of a toluene efflux pump; both Ttg4 and Ttg7 are pyruvate dehydrogenase; Ttg5 is a dihydrolipoamide acetyltransferase; and Ttg7 is the negative regulator of the phosphate regulon. The sequences deduced from ttg8 did not show a significant similarity to any DNA or proteins in sequence databases. Characterization of these mutants and identification of mutant genes suggested that active efflux mechanism and efficient repair of damaged membranes were important in toluene resistance.     

Viral membrane proteins acquire galactose in trans Golgi cisternae during intracellular transport

Frozen, thin sections of baby hamster kidney (BHK) cells were incubated with either concanavalin A (Con A) or Ricinus communis agglutinin I (RCA) to localize specific oligosaccharide moieties in endoplasmic reticulum (ER) and Golgi membranes. These lectins were then visualized using an anti-lectin antibody followed by protein A conjugated to colloidal gold. All Golgi cisternae and all ER membranes were uniformly labeled by Con A. In contrast, RCA gave a uniform labeling of only half to three-quarters of those cisternae on the trans side of the Golgi stack; one or two cis Golgi cisternae and all ER membranes were essentially unlabeled. This pattern of lectin labeling was not affected by infection of the cells with Semliki Forest virus (SFV). Infected cells transport only viral spike glycoproteins from their site of synthesis in the ER to the cell surface via the stacks of Golgi cisternae where many of the simple oligosaccharids on the spike proteins are converted to complex ones (Green, J., G. Griffiths, D. Louvard, P. Quinn, and G. Warren. 1981. J. Mol. Biol. 152:663-698). It is these complex oligosaccharides that were shown, by immunoblotting experiments, to be specifically recognized by RCA. Loss of spike proteins from Golgi cisternae after cycloheximide treatment (Green et al.) was accompanied by a 50% decrease in the level of RCA binding. Hence, about half of the RCA bound to Golgi membranes in thin sections was bound to spike proteins bearing complex oligosaccharides and these were restricted to the trans part of the Golgi stack. Our results strongly suggest that complex oligosaccharides are constructed in trans Golgi cisternae and that the overall movement of spike proteins is from the cis to the trans side of the Golgi stack.     

The SPCA1 Ca2+ Pump and Intracellular Membrane Trafficking

The Golgi apparatus (GA) is a dynamic store of Ca²⁺ that can be released into the cell cytosol. It can thus participate in the regulation of the Ca²⁺ concentration in the cytosol ([Ca²⁺]_cyt), which might be critical for intra‐Golgi transport. Secretory pathway Ca²⁺‐ATPase pump type 1 (SPCA1) is important in Golgi homeostasis of Ca²⁺. The subcellular localization of SPCA1 appears to be GA specific, although its precise location within the GA is not known. Here, we show that SPCA1 is mostly excluded from the cores of the Golgi cisternae and is instead located mainly on the lateral rims of Golgi stacks, in tubular noncompact zones that interconnect different Golgi stacks, and within tubular parts of the trans Golgi network, suggesting a role in regulation of the local [Ca²⁺]_cyt that is crucial for membrane fusion. SPCA1 knockdown by RNA interference induces GA fragmentation. These Golgi fragments lack the cis‐most and trans‐most cisternae and remain within the perinuclear region. This SPCA1 knockdown inhibits exit of vesicular stomatitis virus G‐protein from the GA and delays retrograde redistribution of the GA glycosylation enzymes into the endoplasmic reticulum caused by brefeldin A; however, exit of these enzymes from the endoplasmic reticulum is not affected. Thus, correct SPCA1 functioning is crucial to intra‐Golgi transport and maintenance of the Golgi ribbon.     

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.     

cis-Golgi proteins accumulate near the ER exit sites and act as the scaffold for Golgi regeneration after brefeldin A treatment in tobacco BY-2 cells

The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.     

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.     

Subcompartment localization of the side chain xyloglucan-synthesizing enzymes within Golgi stacks of tobacco suspension-cultured cells

Xyloglucan is the dominant hemicellulosic polysaccharide of the primary cell wall of dicotyledonous plants that plays a key role in plant development. It is well established that xyloglucan is assembled within Golgi stacks and transported in Golgi-derived vesicles to the cell wall. It is also known that the biosynthesis of xyloglucan requires the action of glycosyltransferases including α-1,6-xylosyltransferase, β-1,2-galactosyltransferase and α-1,2-fucosyltransferase activities responsible for the addition of xylose, galactose and fucose residues to the side chains. There is, however, a lack of knowledge on how these enzymes are distributed within subcompartments of Golgi stacks. We have undertaken a study aiming at mapping these glycosyltransferases within Golgi stacks using immunogold-electron microscopy. To this end, we generated transgenic lines of tobacco (Nicotiana tabacum) BY-2 suspension-cultured cells expressing either the α-1,6-xylosyltransferase, AtXT1, the β-1,2-galactosyltransferase, AtMUR3, or the α-1,2-fucosyltransferase AtFUT1 of Arabidopsis thaliana fused to green-fluorescent protein (GFP). Localization of the fusion proteins within the endomembrane system was assessed using confocal microscopy. Additionally, tobacco cells were high pressure-frozen/freeze-substituted and subjected to quantitative immunogold labelling using anti-GFP antibodies to determine the localization patterns of the enzymes within subtypes of Golgi cisternae. The data demonstrate that: (i) all fusion proteins, AtXT1-GFP, AtMUR3-GFP and AtFUT1-GFP are specifically targeted to the Golgi apparatus; and (ii) AtXT1-GFP is mainly located in the cis and medial cisternae, AtMUR3-GFP is predominantly associated with medial cisternae and AtFUT1-GFP mostly detected over trans cisternae suggesting that initiation of xyloglucan side chains occurs in early Golgi compartments in tobacco cells.     

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.     

Cytoplasmic membrane changes in conjugating Tetrahymena

Summary The formation of stacks of smooth-surfaced cisternae was studied in samples of Tetrahymena pyriformis that were prepared for electron microscopy after starvation for 2 days in buffer, and in samples of organisms of different mating type that were starved for 2 days and then mixed to induce conjugation. The number of stacks of cisternae was greater in starved ciliates than in those from stock cultures, and the size and number of stacks increased further after mixing animals of different mating type. When cells were either starved or mixed in buffer to which the protein synthesis inhibitor cycloheximide had been added, the formation of the membranous stacks was almost completely abolished. Addition of the RNA synthesis inhibitor actinomycin D, however, did not result in a significant decrease in the size and number of the stacks of saccules. Conjugation did not occur in the presence of either cycloheximide or actinomycin. The results suggest that protein synthesis is required for the formation of the stacks, but RNA synthesis is no longer necessary for their formation at this stage. The previous identification of the stacks as a Golgi apparatus and the possible functions of these membranes are considered.Supported by grants from NSF (GB-32285) and the American Cancer Society (E-500).The authors acknowledge the technical assistance of Mrs. Sue Thompson.