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The year 1998 is the centenary of the Golgi apparatus. It is an occasion to pay tribute to Camillo Golgi and the other pioneers of Golgi apparatus research. From the very beginning, the discussion of the Golgi apparatus was characterized by a great variety of theories and innovative terminology, with signs of keen interest already evident during the era of light microscopy.
ARF GTPases play a central role in regulating membrane dynamics and protein transport in eukaryotic cells. ARF-like (ARL) proteins are close relatives of the ARF regulators of vesicular transport, but their function in plant cells is poorly characterized. Here, by means of live cell imaging and site-directed mutagenesis, we have investigated the cellular function of the plant GTPase ARL1. We provide direct evidence for a role of this ARL family member in the association of a plant golgin with the plant Golgi apparatus. Our data reveal the existence of key residues within the conserved GRIP-domain of the golgin and within the GTPase ARL1 that are central to ARL1-GRIP interaction. Mutations of these residues abolish the interaction of GRIP with the GTP-bound ARL1 and induce a redistribution of GRIP into the cytosol. This indicates that the localization of GRIP to the Golgi apparatus is strongly influenced by the interaction of GRIP with Golgi-localized ARL1. Our results assign a cellular role to a member of the Arabidopsis ARL family in the plant secretory pathway and propose mechanisms for localization of peripheral golgins to the plant Golgi apparatus.
Golgi apparatus, isolated from rat liver, incorporate [14C]sialic acid from CMP[14C]sialic acid into endogenous glycolipid and glycoprotein acceptors. Incorporation of [14C]sialic acid into endogenous glycoprotein acceptors was stimulated an average of 3-fold by Triton X-100 at an optimal concentration of 0.05% and was inhibited at higher concentrations. Incorporation of [14C]sialic acid into endogenous glycolipid acceptors was not stimulated by detergent. The major glycolipid product was identified by thin-layer chromatography as the ganglioside GD3. SDS-polyacrylamide gel electrophoresis on the glycoprotein products demonstrated incorporation of [14C]sialic acid into 6--7 major bands. Neuraminidase studies determined that approximately 60% of the [14C]sialic acid incorporated into endogenous acceptors in the absence of detergent had a luminal orientation. Furthermore, electron microscopy studies showed that the isolated Golgi apparatus fraction consisted of intact membrane cisternae. Our results demonstrate that sialylation of cisternal acceptors located on the inside of the membrane occurs in the absence of detergent. They are consistent with carrier-mediated transport as a mechanism to allow CMPsialic acid to traverse the Golgi apparatus membrane and to be used to glycosylate endogenous glycoprotein and glycolipid acceptors.
Golgi apparatus isolated from both rat liver and rat kidney have been characterized with respect to their neutral and phospholipid content and their phosphopipid composition and compared with mitochondria, rough endoplasmic reticulum and plasma membranes. In addition, the distribution of sulfatide in the subcellular fractions of rat kidney was determinich are rich in cholesterol esters and ubiquinone. Removal of about 75% of the cisternal contents of rat liver Golgi reduced its content of cholesterol esters but not of ubiquinone. The Golgi complex of liver most closely resembles endoplasmic reticulum in its phospholipid composition except for a higher content of sphingomyelin. Removal of most of the contents of the Golgi cisternae did not appreciably alter the phospholipid composition of the Golgi apparatus of liver. Goligi apparatus from kidney has a phospholipid composition which resembles liver Golgi much more closely than it does any other cell fraction from kidney. The sulfatide content of kidney Golgi, the cell fraction richest in this glycolipid, is about 14% of the total lipid present in this fraction. Sulfatide was present in plasma membranes, mitochondria and rough microsomes, but at about one-third the level found in Golgi. Sulfatide is the main glycosphingolipid present in all the cell fractions from kidney which were studied.
An enzyme that transfers sialic acid from GMP-sialic acid to lactosylceramide was concentrated 40-50 times in Golgi apparatus from rat liver relative to total homogenates. This enzyme required detergents as dispersing agents. Of the numerous detergents tested, the combination Tween 80-Triton CF-54 (1 : 2, w/w) was the most effective in stimulating the reaction. Two apparent pH optima, at 6.35 and 5.5, were observed. The enzyme showed no requirement for a divalent cation. The Km values calculated for CMP-N-acetylneuraminic acid and lactosylceramide were 2.7 - 10(-3) and 1.3 - 10(-4) M, respectively. The enzyme could not be dissociated from Golgi apparatus fractions by treatment with ultrasound, indicating that it is tightly associated with the membrane. The newly synthesized GM3, the product of the reaction, was incorporated into or became tightly associated with the membranes of the Golgi apparatus.
The fate of newly synthesized human immunodeficiency virus type 1 env gp160 was examined in COS-1 cells. The results of morphological chase experiments involving cycloheximide demonstrated that gp160 was retained in the Golgi apparatus for longer than the half-life of the molecule. The degradation of gp160 was insensitive to both bafilomycin A1 and leupeptin (< 0.2 mM), which block lysosomal proteolysis. However, degradation was effectively suppressed by leupeptin at higher concentrations, maximally at 1.7 mM. Furthermore, undegraded gp160 was accumulated in the Golgi apparatus, but was not detected in lysosomes. These results indicate that in COS-1 cells gp160 is not degraded in lysosomes, but rather that degradation takes place in the Golgi apparatus.
We examined the interactions of lipid vesicles (liposomes) labeled with various fluorescent markers with the intracellular membranes of semi-intact ("perforated") fibroblasts. When incubations were performed in the presence of an ATP-regenerating system, both vesicle lipids and entrapped water soluble markers were transferred to the Golgi apparatus of treated cells, indicative of membrane fusion. Fusion occurred using unilamellar vesicles 30-80 nm in diameter and composed of phosphatidylcholine alone, but was inhibited when equimolar amounts of either phosphatidylserine or sphingomyelin were present in the vesicles. Lipid vesicle-Golgi membrane fusion was also inhibited by pretreatment of the perforated cells with N-ethylmaleimide. These findings suggest that lipid vesicles may be useful for delivery of labeled lipids, macromolecules, and dyes to the Golgi apparatus, and for modeling the interactions of transport vesicles with the Golgi apparatus.
Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.
The formation of secretion granules has been studied in the Golgi apparatus of granular epithelial cells of frog urinary bladders maintained at room temperature or cooled at 4 degrees C for various lengths of time. In control animals, the Golgi apparatus was composed of the following stacked elements: subjacent to the cis-element made up of anastomosed tubules, two elements in the mid-compartment consisted of flattened saccules interconnected by tubules. On the trans-face, two or three sacculo-tubular elements were slightly dilated by an electron dense granular material. In the trans-Golgi elements, this material was segregated into dilatations of various sizes and shapes which are continuous with flattened portions devoid of stained material. In the trans-Golgi region, free irregular progranules, seemingly formed by rupture of the trans-most Golgi elements. In granular cells examined after 4 h at 4 degrees C, all Golgi compartments were affected by the low temperature. The cis-half portion of the Golgi apparatus consisted mainly of anastomosed membranous tubules and the cis-element was no longer recognizable. The trans-compartment was reduced to a few flattened saccules with progranules hardly visible on their trans-aspect. At later time intervals, there was a progressive reconstitution of the cis-zone while saccular elements started to pile up in the trans-compartment. At 24 h, the trans-compartment comprised six to eight saccular elements which showed irregular dilatations filled with granular material separated by large flattened portions. These various observations were interpreted as indicating that the trans-compartment was a dynamic structure undergoing continuous renewal.
The nucleus tuberalis lateralis (NTL) and tuberomamillary nucleus (TM), which are located close together in the tuberal region of the human hypothalamus, are differentially affected by Alzheimer's disease (AD), In the AD, the NTL shows only early cytoskeletal alterations, i.e. pre-tangle stages, while the TM is characterised by advanced Alzheimer's changes, e.g. neurofibrillary degeneration, senile plaques and amyloid deposition. Earlier we showed that the early cytoskeletal alterations in the NTL are not accompanied by changes in protein synthetic activity. The present study was carried out in order to measure the protein synthetic activity of the neighbouring area, the TM, which is severely affected by advanced Alzheimer changes. A polyclonal antibody against MG-160, a conserved membrane sialoglycoprotein of the Golgi apparatus, was used to stain this organelle and using an image analysis system, the size of the Golgi apparatus was measured as an index for synthetic and secretory activity in 15 Alzheimer patients and 21 controls. A significant decrease in the size of the Golgi apparatus was found in the TM neurons in AD, although the cell profile area remained unchanged. These data suggest that the protein synthetic and secretory activity of TM neurons is indeed decreased in AD.
Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the Km for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific marker to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.
During all mitotic stages membranous structures can be found around and within the mitotic apparatus of HeLa cells. A careful ultrastructural analysis partly by means of stereo-electron microscopy of semithin (0.5 microns) sections reveals a close association of membranes and microtubules in many regions of the mitotic apparatus. The contact can be direct or via connecting structures (cross-bridges). Referring to various hypotheses concerning the translocation of membranous structures in the cytoplasm of interphase cells (see Suchard and Goode, 1982), the assumption that microtubules participate in the transport of membranous structures in the mitotic apparatus is discussed in detail.
A family of five-pass transmembrane proteins (FinGERs) were identified from the protein sequence database. The family includes yeast Yip1p, Yip4p, Yip5p, and Yif1p, and also their plant, insects, nematode, and mammalian homologues, suggesting their conserved function in a broad range of species. Eight family members were found in human. Multiple sequence alignment revealed three regions conserved among all family members. All of the human family members were expressed widely in various tissues. The human proteins were localized in and around the Golgi apparatus and may also be in the ER to some extent. The Golgi apparatus was fragmented by overexpression of the five of the family members. Some of the members were found to interact by yeast two-hybrid analysis, suggesting the formation of a complex. These results suggest that FinGERs function in maintenance of the Golgi structure and/or transport between the ER and the Golgi apparatus.
Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.
We have dissected the molecular determinants involved in targeting the protein serine kinase PSKH1 to the endoplasmic reticulum (ER), the Golgi apparatus, and the plasma membrane (PM). Given this intracellular localization pattern, a potential role of PSKH1 in the secretory pathway was explored. The amino-terminal of PSKH1 revealed a striking similarity to the often acylated Src homology domain 4 (SH4)-harboring nonreceptor tyrosine kinases. Biochemical studies demonstrated that PSKH1 is myristoylated on glycine 2 and palmitoylated on cysteine 3. Dual amino-terminal acylation targets PSKH1 to Golgi as shown by colocalization with beta-COP and GM130, while nonpalmitoylated (myristoylated only) PSKH1 targets intracellular membranes colocalizing with protein disulphide isomerase (PDI, a marker for ER). Immunoelectron microscopy revealed that the dually acylated amino-terminal domain (in fusion with EGFP) was targeted to Golgi membranes as well as to the plasma membrane (PM), suggesting that the amino-terminal domain provides PSKH1 with membrane specificity dependent on its fatty acylation status. Subcellular fractionation by sucrose gradient analysis confirmed the impact of dual fatty acylation on endomembrane targeting, while cytosol and membrane fractioning revealed that myristoylation but not palmitoylation was required for general membrane association. A minimal region required for proper Golgi targeting of PSKH1 was identified within the first 29 amino acids. Expression of a PSKH1 mutant where the COOH-terminal kinase domain was swapped with green fluorescent protein and cysteine 3 was exchanged with serine resulted in disassembly of the Golgi apparatus as visualized by redistribution of beta-COP and GM130 to a diffuse cytoplasmic pattern, while leaving the tubulin skeleton intact. Our results suggest a structural and regulatory role of PSKH1 in maintenance of the Golgi apparatus, a key organelle within the secretory pathway.
The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.
The plant toxin ricin has to be transported to the Golgi apparatus after endocytosis to exert its toxic effect. In this study we show that transport of apically endocytosed ricin to the Golgi apparatus is stimulated by 8-Br-cAMP in polarized MDCK cells. This stimulation is counteracted by the PKA inhibitor H-89. In contrast, there is no increase in the transport to the Golgi apparatus of ricin internalized from the basolateral membrane. These results suggest that protein kinase A selectively regulates endosome to Golgi transport in these cells.
The hypothesis considers the anatomy and physiology of the memory-storing neuron from the viewpoint that an eccentrically placed nucleus with a Golgi Apparatus situated between it and the proximate cell membrane could be the mechanism for the storage and retrieval of experience and thought processes. Metabolites which enter the Golgi Apparatus are filtered and refined before being discharged from the surface which faces the cell membrane. They travel by the shortest route to a special location on the nearest part of the cell membrane. At this "favoured spot" the action potential is reactivated before the metabolites reach other parts of the membrane. Afferent impulses arriving via the synapses at the "favoured spot" and its tributary dendrites have priority controls the specific polarity of the cell, enabling it to record, store and reproduce the memory of its initial stimulus. Reinforcing stimuli cause the eccentric nucleus and Golgi Apparatus to move nearer to the favoured spot. This mechanism could be the basis of memory. The hypothesis may be applied to other observed phenomenon of brain function and might be submitted to experiment confirmation.