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PubMed2005

Human Yip1A specifies the localization of Yif1 to the Golgi apparatus.

Yip1p and Yif1p are essential for transport from ER to Golgi stack during the early secretory pathway in budding yeast. Here, we report the identification and characterization of human Yif1. Sequence analysis revealed that human Yif1 (HsYif1), like most of the other YIP1 protein family members, contains multiple transmembrane segments. Double immunofluorescence study revealed co-distribution of HsYif1 with Golgi marker such as GS27. To delineate the function of HsYif1, we conducted a yeast two-hybrid assay and identified an interaction between human HsYif1 and HsYip1A, a homolog of yeast Yip1. In addition, our immunoprecipitation pull-down assay validates the interaction between HsYif1 and HsYip1A. Moreover, our immunofluorescence study demonstrates the co-distribution of HsYif1 and HsYip1A. Significantly, over-expression of mutant HsYip1A-lacked cytosolic region disrupts the localization of HsYif1 to the Golgi, suggesting that HsYip1A specifies the localization of HsYif1 to the Golgi. Therefore, we conclude that human Yip1A interacts with and determines the localization of HsYif1 to the Golgi apparatus.

Amino Acid Sequence↗
PubMed2002

FIP-2, an IkappaB-kinase-gamma-related protein, is associated with the Golgi apparatus and translocates to the marginal band during chicken erythroblast differentiation.

Using an antiserum directed against marginal band associated proteins of chicken erythrocytes we isolated clones encoding the chicken homolog of 14.7K-interacting protein 2 (FIP-2), a protein potentially involved in tumor necrosis factor-alpha/nuclear factor-kappaB signaling, from a chicken erythroblast cDNA library. We found that chicken FIP-2 was expressed in a variety of tissues and cell types, but unlike its human counterpart, alternative splicing does not appear to take place. Analysis of intracellular localization revealed that FIP-2 was concentrated at the Golgi apparatus in most cells. Perturbation of the Golgi structure without loss of Golgi function (by treatment with nocodazole) resulted in a retention of FIP-2 at the dispersed Golgi fragments. In contrast, disruption of both Golgi structure and function (by brefeldin A) led to a loss of FIP-2 from Golgi membranes. Remarkably, during erythroblast differentiation FIP-2 was found to translocate from the Golgi to the marginal band. Our results support the hypothesis of a function of the Golgi apparatus in signal transduction. Moreover, our results raise the possibility that the marginal band of chicken erythrocytes, in addition to its role in morphogenesis, has a function in signal transduction and that FIP-2 is in some way involved in its formation.

Amino Acid Sequence↗
PubMed1999

Palmitoylation of GAP-43 by the ER-Golgi intermediate compartment and Golgi apparatus.

Palmitoylation of the neuronal plasticity protein GAP-43 has previously been shown to occur at the plasma membrane, but the site of initial palmitoylation has not been identified. To identify this organelle we have incubated GAP-43 with various subcellular fractions and have analyzed palmitoylation by the Triton X-114 partitioning method. In vitro-translated [(35)S]methionine-labeled GAP-43 was incubated with plasma membrane, nuclei, mitochondria, Golgi apparatus and a rough microsome preparation that contained the ER-Golgi intermediate compartment (ERGIC), but not plasma membrane or Golgi apparatus. GAP-43 partitioned into Triton X-114 in the presence of plasma membrane, Golgi, and ERGIC membranes, but not nuclei or mitochondria. Partitioning caused by the ERGIC was blocked by pretreatment of the membranes with the palmitoylation inhibitors dithiothreitol, tunicamycin, and low temperature, and by treatment of GAP-43 with iodoacetamide. The time course of partitioning agreed closely with the time course of incorporation of radioactive palmitate into proteins as reported previously. Because the ERGIC has a broad distribution in the cell, our results provide evidence that the ERGIC is the initial site of GAP-43 palmitoylation.

Acylation↗
PubMed1998

Localization of Tctex-1, a cytoplasmic dynein light chain, to the Golgi apparatus and evidence for dynein complex heterogeneity.

To date, much attention has been focused on the heavy and intermediate chains of the multisubunit cytoplasmic dynein complex; however, little is known about the localization or function of dynein light chains. In this study, we find that Tctex-1, a light chain of cytoplasmic dynein, localizes predominantly to the Golgi apparatus in interphase fibroblasts. Immunofluorescent staining reveals striking juxtanuclear staining characteristic of the Golgi apparatus as well as nuclear envelope and punctate cytoplasmic staining that often decorates microtubules. Tctex-1 colocalization with Golgi compartment markers, its distribution upon treatment with various pharmacological agents, and the cofractionation of Tctex-1-associated membranes with Golgi membranes are all consistent with a Golgi localization. The distribution of Tctex-1 in interphase cells only partially overlaps with the dynein intermediate chain and p150(Glued) upon immunofluorescence, but most of Tctex-1 is redistributed onto mitotic spindles along with other dynein/dynactin subunits. Using sequential immunoprecipitations, we demonstrate that there is a subset of Tctex-1 not associated with the intermediate chain at steady state; the converse also appears to be true. Distinct populations of dynein complexes are likely to exist, and such diversity may occur in part at the level of their light chain compositions.

Amino Acid Sequence↗
PubMed2000

Inositol 1,4,5-trisphosphate but not ryanodine-receptor agonists induces calcium release from rat liver Golgi apparatus membrane vesicles.

We investigated the direct effect of inositol 1,4,5-trisphosphate (IP(3)) and ryanodine receptor agonists on Ca(2+) release from vesicles of a rat liver Golgi apparatus (GA) enriched fraction, which were actively loaded with (45)Ca(2+). Results in GA were compared with those obtained in a rat liver endoplasmic reticulum (ER) enriched fraction. The addition of IP(3) at concentrations ranging from 100 nm to 100 microm, in the presence of thapsigargin, a specific inhibitor of sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPases, promoted a rapid decrease in the Ca(2+) content of GA vesicles. The amount of Ca(2+) released from the vesicles was a function of IP(3) concentration, reaching about 60% in both GA and ER fractions at 100 microm IP(3). Calcium release was inhibited by heparin, an antagonist of IP(3) receptors. Calcium exhibited a bell-shaped effect on IP(3)-dependent Ca(2+) released from GA vesicles: it activated Ca(2+) release at concentrations up to 1 microm, and inhibited it at higher concentrations. In contrast to that found in the endoplasmic reticulum fraction, none of the ryanodine receptor agonists tested (cyclic ADP-ribose, caffeine and ryanodine) significantly induced Ca(2+) release from GA fraction vesicles in the presence of thapsigargin. Our results indicate the presence of an IP(3)-sensitive Ca(2+) release mechanism in the Golgi apparatus membrane analogous to that of the ER. However, a Ca(2+) release mechanism sensitive to ryanodine receptor agonists like that of ER is not evident in the GA membrane.

Adenosine Diphosphate Ribose↗
PubMed1998

Effect of ethanol administration on the level of dolichol in rat liver microsomes and Golgi apparatus.

Data obtained in our laboratory had suggested that acute ethanol administration (6 g/kg body weight) selectively and rapidly affects the intracellular system of protein glycosylation at the level of the Golgi apparatus. Dolichols are important membrane components, and dolichyl phosphate is a glycosyl sugar carrier for N-glycosylation of proteins in endoplasmic reticulum and is considered rate-limiting for this process. In this study, modifications in the concentration and distribution of liver microsomal dolichols after acute ethanol administration were investigated. Between 3 and 24 hr after ethanol administration, the microsomal dolichyl phosphate concentration was significantly lower than in control animals. The highest reduction was observed at 12 hr (-52%). An earlier and more marked reduction of total dolichol was observed in the Golgi apparatus, and, in particular, in the secretory fraction F1 (-70% at 6 hr). Ethanol treatment of isolated hepatocytes led to a significant reduction of the de novo synthesis of both dolichyl phosphate and free dolichol. Moreover, in vitro experiments have demonstrated that pro-oxidant agents lead to a significant decrease of both free dolichol and dolichyl phosphate. Our results suggest that acute ethanol administration induces a marked decrease of dolichols, probably by increasing the degradation and impairing the biosynthetic pathway of these molecules.

Animals↗
PubMed2005

Agonist-induced vesiculation of the Golgi apparatus in pancreatic acinar cells.

BACKGROUND & AIMS: The pancreatic acinar cell is known to regulate exocytosis, total protein synthesis, and secretory protein transport in response to a secretory stimulus. Whether secretory vesicle formation also is regulated is unclear. In this study, we determined whether agonist stimulation induces morphologic alterations in the acinar cell Golgi apparatus, and we evaluated the role of the vesicle severing protein dynamin. METHODS: Changes in Golgi structural integrity by examining the distribution of various Golgi and TGN lipid and protein markers in live and fixed cells on stimulation with cholecystokinin were noted in a primary pancreatic acinar cell model. Multiple dynamin reagents were used to examine the distribution and function of this molecular pinchase in resting and stimulated cells. RESULTS: Regulated secretion in acinar cells induced (1) marked fragmentation of the trans-Golgi network (TGN) that corresponded temporally with an increase in cytoplasmic calcium whereas pre-TGN compartments of the Golgi and regions of the TGN involved in the generation of constitutively trafficking vesicles were unaffected by agonist, and (2) significant recruitment of dynamin to the acinar cell Golgi apparatus that appeared to potentiate fragmentation of the TGN. CONCLUSIONS: These results suggest that the TGN is a dynamic organelle that fragments in response to cholecystokinin stimulation, a process that may contribute to zymogen granule formation.

Animals↗
PubMed1998

Conservation of the architecture of the Golgi apparatus related to a differential organization of microtubules in polykaryocytes induced by syn- mutants of herpes simplex virus 1.

Infection of Vero and HEp-2 but not of 143TK- cells with herpes simplex virus 1 results in fragmentation and dispersal of the Golgi apparatus. Concurrently, in all three infected cell lines the microtubular network is disrupted, suggesting that the disruption of microtubules is essential but not sufficient to induce the fragmentation of the Golgi apparatus. We now report the following: (i) In polykaryocytes formed in Vero cells infected with HSV-1 syn- mutant viruses, intact Golgi stacks were readily detected by electron microscopy. These aggregated in the center of large polykaryocytes. (ii) The distribution of viral glycoprotein D, examined in both fixed and nonfixed cells, appeared to match the distribution of the Golgi stacks, suggesting that the aggregated Golgi stacks funnel viral glycoproteins and viral particles to a limited region of the plasma membrane of the polykaryocytes rather than directing exocytic flow in a more dispersed fashion as seen in syn+ virus-infected cells exhibiting fragmented and dispersed Golgi. (iii) In most polykaryocytes, the microtubules formed parallel bundles extending along the axis of recruitment of new cells. (iv) Fragmentation of the microtubules at the periphery of the cell near the plasma membrane was observed in untreated or cycloheximide-treated cells 2 h after infection with syn- virus HSV-1(MP) or syn+ HSV-1(mP) but not in mock-infected cells. These observations suggest that peripheral depolymerization is initiated at the time of infection and that a factor which determines the syn- or syn+ phenotype is whether the microtubular network regenerates concomitant with cell fusion or reorganizes to form a collapsed network surrounding nuclei of syn+ infected cells.

Animals↗
PubMed1997

Ultrastructural changes in the Golgi apparatus and secretory granules of HL-60 cells treated with the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin inhibits the N-linked oligosaccharide processing enzymes alpha-glucosidases I and II, and the ceramide specific glucosyltransferase which catalyses the first step in glucosphingolipid biosynthesis. We have studied the effects of this compound on the ultrastructure of HL-60 cells to identify novel activities of this compound. Treatment of HL-60 cells with this imino sugar results in several morphological changes within the cell, none of which result in cytotoxicity. The plasma membrane stains heavily with potassium ferrocyanide within 30 min following addition of the compound to the medium, and there is then a time dependent involvement of all other intracellular membranes. Secretory granules become enlarged and lose their dense core morphology and appear either empty and vacuolated or have low density contents. However, the most striking effect of NB-DNJ treatment is on the Golgi apparatus. The Golgi exhibits a time-dependent change from typical Golgi morphology to a structure almost completely devoid of cisternae and consisting predominantly of vesicles. All the observed changes are fully reversible on withdrawal of the compound.

1-Deoxynojirimycin↗
PubMed2006

The mitotic phosphatase cdc25C at the Golgi apparatus.

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity of cdc25C is regulated by phosphorylation which by itself is implicated in regulating the subcellular localization. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures which by immunofluorescence analysis as well as by biochemical subfractionation turned out to be the Golgi apparatus. It will be further shown that cdc25C at the Golgi fraction is an active phosphatase suggesting an additional and new role of cdc25C at the Golgi apparatus.

Cell Cycle Proteins↗
PubMed2006

Fragmentation of the Golgi apparatus in neurodegenerative diseases and cell death.

Fragmentation of the neuronal Golgi apparatus (GA) was reported in amyotrophic lateral sclerosis (ALS), corticobasal degeneration, Alzheimer's and Creutzfeldt-Jacob disease, and in spinocerebelar ataxia type 2 (SCA2). In transgenic mice expressing the G93A mutant of Cu/Zn superoxide dismutase (SOD1) of familial ALS (fALS), fragmentation of the GA of spinal cord motor neurons and aggregation of mutant protein were detected months before the onset of paralysis. Moreover, cells that expressed the G93A and G85R mutants of SOD1 showed fragmentation of the GA and decreased viability without apoptosis. We summarize here mechanisms involved in Golgi fragmentation implicating: (a) the dysregulation by mutant SOD1of the microtubule-destabilizing protein Stathmin, (b) the disruption by mutant SOD1of the neuronal cytoplasmic dynein, (c) the coprecipitation of mutant SOD1 with Hsp25 and Hsp27, (d) the reduction of detyrosinated microtubules by aggregated tau which resulted in non-apoptotic cell death and (e) the disruption by mutant growth hormone of the trafficking from the rough endoplasmic reticulum to the GA. The data indicate that neuronal Golgi fragmentation is an early and probably irreversible lesion in neurodegeneration, caused by a variety of mechanisms. Golgi fragmentation is not secondary to apoptosis but it may "trigger" apoptosis.

Cell Death↗
PubMed1970

Identity of very low density lipoprotein apoproteins of plasma and liver Golgi apparatus.

In the rat, very low density lipoproteins isolated from hepatocyte Golgi apparatus, liver perfusates, and whole plasma appear identical in many respects. With specific immunochemical techniques and polyacrylamide-gel electrophoresis it can be demonstrated that the very low density lipoproteins from all three sources contain the same major lipoprotein apoproteins.

Animals↗
PubMed1989

A novel 58-kDa protein associates with the Golgi apparatus and microtubules.

With the aim of identifying proteins involved in linking microtubules to other cytoplasmic structures, microtubule-binding proteins were isolated from rat liver extracts by a taxol-dependent procedure. The major non-tubulin component, a 58-kDa protein (designated 58K), was purified to homogeneity by gel filtration chromatography. To aid further characterization of 58K, purified preparations of the protein were used as immunogen for the production of monoclonal antibodies. Five different monoclonals were obtained, and each of these reacted on immunoblots of liver homogenates with a single band that comigrated with 58K. Based on the results of immunochemical, peptide mapping, and microsequencing experiments, 58K was found to be unrelated structurally to similarly sized cytoskeleton-associated proteins, such as tubulin, tau, vimentin, or keratin, and to represent a new protein species. Several in vitro properties of 58K were found to be characteristic of microtubule-associated proteins. For instance, 58K cosedimented quantitatively with microtubules out of liver extracts, stimulated polymerization of tubulin, and bound to microtubules in a saturable manner. In contrast to traditional microtubule-associated proteins, however, 58K was not found to be distributed uniformly along microtubules in cells. Immunofluorescence microscopy of cultured hepatoma cells revealed, instead, that 58K is associated principally with the Golgi apparatus. Moreover, Golgi membranes isolated from rat liver were observed by immunoblotting to contain significant levels of 58K, which, upon subfractionation of the membranes, partitioned as if it were a peripheral membrane protein exposed to the cytoplasmic side of the Golgi. These collective results have been evaluated in terms of earlier evidence that the intracellular position and structural integrity of the Golgi relies on the presence and organization of microtubules. In that context, the observations reported here suggest that the in vivo function of 58K is to provide an anchorage site for microtubules on the outer surface of the Golgi.

Animals↗
PubMed1991

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.

Animals↗
PubMed1978

Localization of somatostatin-like immunoreactivity in the Golgi apparatus of central and peripheral neurons.

With the indirect immunofluorescence technique of Coons and collaborators somatostatin-like immunoreactivity (SLI) was observed in certain neurons of the central and peripheral nervous system of the rat. In the cell bodies a strong SLI was observed with a distribution resembling that of the Golgi apparatus. In addition a weak SLI was diffusely distributed in the cytoplasm. After photography the sections processed for immunocytochemistry were stained with the thiamine pyrophosphatase technique of Novikoff and Goldfischer. The latter technique is assumed to be a specific marker for the Golgi complex. It was found that the strong SLI and the thiamine pyrophosphatase activity had an identical distribution. Thus, one pool of somatostatin appears to be localized to the Golgi apparatus.

Animals↗
PubMed1983

The rough endoplasmic reticulum and the Golgi apparatus visualized using specific antibodies in normal and tumoral prolactin cells in culture.

Antibodies directed against membrane components of dog pancreas rough endoplasmic reticulum (A-RER) and rat liver Golgi apparatus (A-Golgi) (Louvard, D., H. Reggio, and G. Warren, 1982, J. Cell Biol. 92:92-107) have been applied to cultured rat prolactin (PRL) cells, either normal cells in primary cultures, or clonal GH3 cells. In normal PRL cells, the A-RER stained the membranes of the perinuclear cisternae as well as those of many parallel RER cisternae. The A-Golgi stained part of the Golgi membranes. In the stacks it stained the medial saccules and, with a decreasing intensity, the saccules of the trans side, as well as, in some cells, a linear cisterna in the center of the Golgi zone. It also stained the membrane of many small vesicles as well as that of lysosomelike structures in all cells. In contrast, it never stained the secretory granule membrane, except at the level of very few segregating granules on the trans face of the Golgi zone. In GH3 cells the A-RER stained the membrane of the perinuclear cisternae, as well as that of short discontinuous flat cisternae. The A-Golgi stained the same components of the Golgi zone as in normal PRL cells. In some cells of both types the A-Golgi also stained discontinuous patches on the plasma membrane and small vesicles fusing with the plasma membrane. Immunostaining of Golgi membranes revealed modifications of membrane flow in relation to either acute stimulation of PRL release by thyroliberin or inhibition of basal secretion by monensin.

Animals↗
PubMed1985

UDPglucose-ceramide glucosyltransferase from porcine submaxillary glands is associated with the Golgi apparatus.

Subcellular distribution of pig submaxillary gland UDPglucose-ceramide glucosyltransferase (EC 2.4.1.80), the enzyme which catalyses the first step during the sequential addition of carbohydrate moieties for ganglioside biosynthesis, was studied. The results presented strongly suggest that in pig submaxillary gland, the transfer of glucose on endogenous or exogenous ceramides takes place in the Golgi apparatus: the specific activity of UDPglucose-ceramide glucosyltransferase increased in parallel with the activity of a known marker of the Golgi apparatus, UDPgalactose-ovomucoid galactosyltransferase. The specific activity of the glucosyltransferase was 18-times higher in the purified Golgi membranes than in the postnuclear supernatant and the yield was over 30%. An apparent Km of 22 microM for UDPglucose and 54 microM for ceramides was determined. Maximal glucosylation of endogenous ceramides was achieved at pH 6.5 in the presence of NADH (1 mM) as inhibitor of pyrophosphatases and with Mn2+ (5 mM). It was found that the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) is an efficient activator for the glucosylation of exogenous ceramides.

Animals↗
PubMed1995

Dynamic structure and function of Golgi apparatus in the salivary acinar cells.

The structural dynamics of the Golgi apparatus (GA) in salivary exocrine cells under various conditions are described. The GA of a normal acinar cell is a single continuous structure with a stack of 4-5 cisternae associated with small vesicles 50 to 70 nm in diameter, and vacuoles of varying diameter. There is sufficient indication that the structural integration of the GA into one single organelle is maintained by the effect of microtubules. Disorganization of the lamellated structure was recognized in host cells under various conditions such as mitotic division and treatment with monensin, brefeldin A or okadaic acid. Perturbation of the transport of exportable proteins appeared to be a major factor of the structural disorganization. Structural analysis during the process of recovery from the effects of these drugs could provide useful model to examine the controlling mechanism of morphogenesis of GA. Based on these observations and related evidences, this review article discusses the characteristic lamellated structure of the GA to be essential for the expression of normal functions.

Animals↗