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PubMed1986

Mutations blocking the transport of the influenza virus hemagglutinin between the rough endoplasmic reticulum and the Golgi apparatus.

Mutants ts1 and ts227 of fowl plague virus have a temperature-sensitive defect in the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus. The primary structure of the hemagglutinin of the mutants and of a number of revertants derived from them has been analysed by nucleotide sequencing. The transport block of the hemagglutinin of ts227 can be attributed to a single amino acid exchange. It involves the replacement of aspartic acid at position 457 by asparagine thereby introducing a new glycosylation site which appears to be located in a cryptic position in the lower part of the hemagglutinin stalk. Attachment of carbohydrate to this site is temperature-dependent. At permissive temperature only a small fraction of the monomers (approximately 30%) is glycosylated in this position, whereas at nonpermissive temperature this is the case with all subunits. The data suggest that under the latter conditions the new oligosaccharide interferes by steric hindrance with the trimerization of the hemagglutinin. The hemagglutinin of ts1 has an essential amino acid exchange at position 275 where serine is replaced by glycine. This substitution may increase the flexibility of the molecule in the hinge region between the globular domain and the stalk. The exchange of a conserved glutamic acid residue at position 398 that is involved in the interaction between different monomers contributes also to the structural instability of the ts1 hemagglutinin. These observations support the notion that the transport of the hemagglutinin from the rough endoplasmic reticulum to the Golgi apparatus depends on trimer assembly.

Animals↗

PubMed1994

The functioning of the yeast Golgi apparatus requires an ER protein encoded by ANP1, a member of a new family of genes affecting the secretory pathway.

Mnt1p is an alpha 1.2-mannosyltransferase which resides in an early compartment of the Saccharomyces cerevisiae Golgi apparatus. We have shown that the signal-anchor region is sufficient, and the transmembrane domain necessary, for its normal Golgi localization. This is similar to the transmembrane domain-mediated retention of mammalian glycosyltransferases, and distinct from the tail-mediated recycling retention of certain mammalian and yeast trans-Golgi proteins. To examine the mechanism involved in transmembrane domain-mediated retention, we have isolated six classes of mutants which fail to retain Mnt1p-reporter fusions in the early Golgi. These mutants all show additional phenotypes which are consistent with alterations in Golgi function. We have called the mutant classes 'gem', for Golgi enzyme maintenance. GEM3 is identical to the previously cloned gene ANP1, and homologous to VAN1 and MNN9. Together, these define a new class of proteins involved in the organization and functioning of the secretory pathway. Interestingly, Anp1p is localized to the endoplasmic reticulum (ER), implying that some function of the ER is required to maintain a functional Golgi apparatus.

Amino Acid Sequence↗

PubMed2003

Ubiquitin-dependent and -independent proteasomal degradation of apoB associated with endoplasmic reticulum and Golgi apparatus, respectively, in HepG2 cells.

Studies in hepatocyte cultures indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the proteasome pathway is a major mechanism for the degradation. In the present study, we have examined the detailed itinerary of apoB degradation through its secretory pathway in HepG2 cells. We found that ubiquitin-dependent proteasomal degradation of apoB largely occurred on the cytosolic surface of rough and smooth endoplasmic reticulum (ER) and that a small proportion of apoB was dislodged from the secretory organelles into the cytosolic compartment where it underwent ubiquitination for proteasomal degradation. The transmembrane conformation of apoB persisted as the protein was transported through the Golgi apparatus. We further demonstrated that proteasomal degradation of apoB was associated the Golgi apparatus but Golgi-associated apoB was not ubiquitinated, indicating an ubiquitin-independent proteasomal degradation of apoB is associated with this organelle. We conclude that apoB undergoes proteasomal degradation while going through different compartments of the secretory pathway; further, ER-associated proteasomal degradation of apoB in the ER is ubiquitin-dependent whereas that occurring in the Golgi is ubiquitin-independent.

Apolipoprotein B-100↗

PubMed1991

Cytochemical characteristics of the Golgi apparatus.

Lectinocytochemistry provides a useful tool for localizing subcompartments of the complex reticular apparatus of Golgi. The technique is based on interactions of lectins with glycoconjugates present in the limiting membranes and luminal spaces of Golgi elements. Application of a series of lectins of different sugar specificities permits a differentiation between Golgi subcompartments containing glycoconjugates with different oligosaccharide side chains. These may be a) differnet glycoconjugates or b) glycoconjugates at different stages during synthesis or repair of their glycans. The lectinocytochemical studies with mannose-, glucose-, N-acetyl-glucosamine-, N-acetyl-galactosamine-, galactose-, fucose-, and sialic acid-recognizing lectins revealed predominating patterns that labeled distinct, i.e., cis, medial, trans, and transmost, regions of the Golgi apparatus. A further refinement could be achieved by differential lectin-inhibition that enables a dissection of lectin binding reactions on the basis of their binding affinities. High-affinity binding reactions showed that subcompartments are not necessarily confined to one single Golgi subregion and may change their position from one to another subregion. Some of the patterns observed may be interpreted in relation to certain steps during synthesis and modifications of glycans.

Animals↗

PubMed1995

Distribution of Sertoli cell microtubules, microtubule-dependent motors, and the Golgi apparatus before and after tight junction formation in developing rat testis.

Sertoli cells are polarized epithelial cells of the seminiferous epithelium which provide structural and physiological support for differentiating germ cells. They establish different basal and adluminal environments for the selective nurturing of pre- and post-meiotic germ cells within the seminiferous epithelium, segregated by the Sertoli-Sertoli cell tight junctional complex, the blood-testis barrier. Tight junction formation between epithelial cells in vitro is a critical polarizing event associated with changes in polarized targeting of membrane-specific proteins and reorganization of microtubules, centrioles, and the Golgi apparatus. To investigate whether tight junction formation is associated with organelle reorganization in Sertoli cells in vivo, we have characterized distribution patterns of Sertoli cell microtubules, the mechanoenzymes kinesin and cytoplasmic dynein, and the Golgi apparatus during tight junction formation in developing rat testis. Immunocytochemistry on samples taken at 5, 10, 15, 20, and 25 days of age was used to examine the distribution of these proteins during the extensive cellular reorganization that culminates in the formation of the blood-testis barrier at 19 days of age. Our data show that the distribution patterns reflect the extensive intercellular repositioning of tubule cells in developing seminiferous tubules, but that changes in intracellular organization are not temporally associated with formation of the blood-testis barrier.

Age Factors↗

PubMed1977

Localization of some glycolipid glycosylating enzymes in the Golgi apparatus of rat kidney.

Cell fractions from rat kidney were isolated and studied for their ability to synthesize several possible intermediates in the biosynthesis of sulfatides and gangliosides. The enzymes studied include UDP-Gal:ceramide galactosyltransferase, UDP-Gal:glucosylceramide galactosyltransferase, UDP-Gal:galactosylceramide galactosyltransferase, and CMP-NAN:lactosylceramide sialytransferase activities. The initial glycosylation of ceramide was found to be present in all of the kidney cell fractions studied. The remaining glycosylating enzymes were largely localized in the Golgi apparatus of kidney. Thus, in addition to modifying glycoproteins for secretion, the Golgi apparatus in kidney is involved in the modification of a number of glycolipids which are destined to form cell membrane components.

Animals↗

PubMed2002

Cloning of the cDNA and mRNA expression of CLRP, a complex leucine repeat protein of the Golgi apparatus expressed by specific neurons of the rat brain.

We report the molecular cloning of one novel cDNA isolated from the rat brain. We have named the putative protein CLRP, for complex leucine-repeat protein. The predicted CLRP amino acid sequence shares homology in the amino acid composition with the Galactose, N-Acetylglucosamine, and Sialic acid transporters, and shows 91% identity with the sequence of one human chromosome 5 BAC clone. Expression of the CLRP cDNA tagged with GFP in COS-7 cells was found in cell organelles that resemble the Golgi apparatus of the cytoplasm. In Northern blot, the CLRP probe labels a single band of 2.4 kb in the brain, kidney, lung, testis, and prostate. In the brain, CLRP mRNA is expressed by limited sets of neurons, such as the pyramidal cells of the cortex, the Purkinje cells of the cerebellum, and the motoneurons of the brainstem. In the brain, the CLRP mRNA is expressed at embryonic day 15; levels of expression are maintained until postnatal day 10 and decrease in adults. The results suggest that CLRP codes a novel member of the nucleotide-sugar family of proteins of the Golgi apparatus.

Animals↗

PubMed1989

Involvement of the Golgi apparatus in sorting of materials to opposite ends of frog rod retinal photoreceptors.

We have studied the rod cells of retinas of Rana pipiens by phosphatase cytochemistry and immunocytochemistry. We find that the Golgi apparatus of these cells, although different in its intracellular distribution from that of other neurons, has a cis-trans organization like that of other neurons as regards morphological features and the distribution of phosphatase activities. Antibodies against opsin bind to several sacs of the rod Golgi apparatus, especially those at the trans side of the Golgi stack. This suggests that Golgi involvement in the packaging of opsin for eventual delivery to the photoreceptive outer segments of the cell involves passage through trans Golgi systems. Proteins destined for the opposite end of the cell--the presynaptic terminal--also seem to pass through trans Golgi systems, as is indicated both by immunocytochemical localization of the synaptic vesicle protein p38 (synaptophysin) and by the presence of thiamine pyrophosphatase activity in some of the synaptic vesicles. Our findings suggest that sorting of membrane proteins destined for opposite ends of the photoreceptor takes place in systems at or near the trans Golgi face.

Animals↗

PubMed1994

Inhibition of protein synthesis disrupts the Golgi apparatus in the fission yeast, Schizosaccharomyces pombe.

Schizosaccharomyces pombe was treated with either cycloheximide or anisomycin at levels sufficient to inhibit > 95% of protein synthesis for periods upon to 3 h, equivalent to one cell cycle. Treatment for as little as 1 h caused significant loss of the Golgi apparatus by both immunofluorescence and electron microscopy. The loss was quantitated by stereology on electron micrographs. Nearly 90% of the stacked Golgi was lost over a 3 h period. No other intracellular membrane compartment seemed to be affected. Measurement of enzyme activities confirmed these observations. The activity of a resident of the Golgi apparatus, alpha-1,2 galactosyltransferase, was reduced over this time, whereas the endoplasmic reticulum marker, BiP, and the cytoplasmic enzyme, hexokinase, were unaffected. The morphological changes associated with cycloheximide addition were reversed on its removal, though there was a lag before cells recommenced growth or secretion of the enzyme, acid phosphatase.

Acid Phosphatase↗

PubMed1996

Enzyme-specific transport of rat liver cytochrome P450 to the Golgi apparatus.

It has been reported that cytochrome P450 is expressed in the plasma membrane of hepatocytes isolated from human and rat. Cytochrome P450s expressed on the cell surface are potential targets for the immune response of drug-induced and autoimmune hepatitis. However, the mechanisms behind transport of cytochrome P450 to the plasma membrane are obscure. The present investigation aimed at identifying cytochrome P450 expressed in the Golgi apparatus. Golgi membrane fractions from rat liver were prepared and characterized: one enriched with cis-Golgi, one highly enriched with trans-Golgi, and one intermediate Golgi fraction representing medial-Golgi. In these three fractions, significant amounts of cytochrome P450 and NADPH cytochrome P450 reductase were present, which could not be accounted for by contamination with endoplasmic reticulum. A marked difference between the relative content of different cytochrome P450 enzymes was found. CYP4A1 was found at the highest concentration, CYP2E1 at an intermediary level, and CYP1A2 at low levels, whereas no Golgi-specific CYP3A1 was detectable. It was also shown that the CYP2E1 present in the Golgi fractions was catalytically active. It is suggested that various forms of hepatic cytochrome P450 are transported to the plasma membrane through the Golgi apparatus in an enzyme-specific manner.

Animals↗

PubMed1997

Phosphorylation of osteopontin by Golgi apparatus casein kinase.

Osteopontin (OPN) is a ubiquitous multiphosphorylated secretory glycoprotein. Twenty-seven phosphorylated serines have been identified in bovine milk OPN (E. S. Sorensen et al. (1995) Protein Sci. 4, 2040-2049). Nineteen of these phosphoacceptor sites are fully conserved in rat OPN, all displaying the consensus for the Golgi apparatus casein kinase, G-CK (S-x-E/Sp). Here we show that rat OPN is indeed phosphorylated more readily than casein itself by G-CK from either rat mammary gland or liver. OPN is also phosphorylated by casein kinases-1 and -2 (CK1, CK2), though less readily than casein. If OPN kinase activities are normalized in terms of casein phosphorylation, OPN phosphorylation rate by G-CK is 78-fold and 19-fold higher than those measured with CK2 and CK1, respectively. These data, in conjunction with the specific location of G-CK to the Golgi apparatus, where CK2 and CK1 are hardly detectable, support the view that G-CK is the main if not the only physiological agent committed to the phosphorylation of OPN.

Amino Acid Sequence↗

PubMed2002

The cleavage plane will bend when one aster of the mitotic apparatus stops growing in compressed sea urchin eggs.

Astral microtubules are elongated greatly during anaphase and telophase in sea urchin eggs. The surface density of microtubules reaching the cell surface can be defined at each surface point. Gradients of the surface-density function were assumed to drive membrane proteins whose accumulation causes the formation of contractile-ring microfilaments. An equation was constructed to calculate the movements of the membrane proteins on a curved surface. The equation was applied to eggs compressed between a coverslip and a glass slide by regarding the egg shape as an oblate spheroid. The simulations explained the observations that contractile-ring microfilaments locally appeared and then developed into a complete ring in compressed eggs. When one aster in the mitotic apparatus stopped growing during anaphase, the equation predicted that the zone of contractile-ring microfilaments is displaced toward the inactivated aster, curves in the view from above and tapers off toward the cell edge. The curve gets sharper as eggs are compressed more greatly and as microtubules from the growing aster penetrate more deeply into the opposite hemisphere. The predictions were compared with the observations by Ishii and Shimizu in 1995 and by Hamaguchi in 1998 regarding the furrow formation by the asymmetric mitotic apparatus.

Actin Cytoskeleton↗

PubMed1995

Immunolocalization of the heterotrimeric kinesin-related protein KRP(85/95) in the mitotic apparatus of sea urchin embryos.

We have used monoclonal antibodies to perform confocal light microscopic immunolocalization of KRP(85/95), a heterotrimeric plus-end-directed microtubule motor protein, in dividing cells of sea urchin embryos. Embryos were stained during the first division cycle, and dissociated blastomeres were stained at the 32- to 64-cell stages. Double labeling of the dividing cells with anti-tubulin and anti-KRP(85/95) showed a clear concentration of the motor protein in the mitotic apparatus; KRP(85/95) appeared to associate with pericentriolar regions during prophase, with kinetochore-to-pole microtubules during metaphase, and, in a striking fashion, with the spindle interzone during anaphase. KRP(85/95) began to accumulate in the interzone immediately following chromosome separation and the area of concentration expanded with the lengthening of the interzonal region during anaphase. During telophase KRP(85/95) appeared to disperse with the establishment of the cleavage furrow and did not concentrate in the midbody. KRP(85/95) staining in the mitotic apparatus was punctate and detergent-sensitive, suggesting an association with membranous vesicles, but unlike kinesin, KRP(85/95) did not appear to codistribute with calsequestrin-containing endoplasmic reticulum. Finally, KRP(85/95) appears to be present in dividing blastomeres up to at least the blastula stage, but, unlike kinesin, it is not expressed in terminally differentiated, nonmitotic coelomocytes of the adult animal. These results suggest that the expression and targeting of KRP(85/95) and kinesin differ and that KRP(85/95) may play a role in vesicle transport during embryonic cell division.

Animals↗

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↗

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↗

PubMed1993

Membrane cycling between the ER and Golgi apparatus and its role in biosynthetic transport.

Membrane traffic between the ER and Golgi is now recognized as a carefully regulated process controlled by distinct anterograde (to the Golgi) and retrograde (to the ER) pathways. These pathways link two organelles with different morphologies, structures, and localizations within the cell. The ER, which is involved in multiple cellular functions including protein biosynthesis and folding, extends to the cell periphery and forms a dynamic tubule reticulum. By contrast, the Golgi apparatus, which functions in membrane sorting and recycling events, is localized at the center of the cell near the MTOC and is comprised of compact cisternal units. The required transport into the Golgi apparatus of newly synthesized proteins exported from the ER offers a twofold advantage to the cell. First, the rate of movement of membrane and protein through the biosynthetic pathway can be controlled by the selective use of a recycling pathway. Second, membrane moving through the biosynthetic pathway enters a structure specialized for sorting of membrane to different final destinations in the cell Control of biosynthetic transport within the ER/Golgi system involves the utilization of two alternative transport pathways: anterograde (ER to Golgi) and retrograde (Golgi to ER). These two pathways share a common regulatory system involving membrane assembly/disassembly of cytosolic coatomer proteins. Thus, conditions that favor irreversible coatomer binding (i.e., GTP gamma S) inhibit retrograde transport while producing anterograde transport intermediates. Conditions that prevent coatomer binding (i.e., BFA) inhibit anterograde transport and enhance retrograde transport. The underlying biochemical machinery that normally balances anterograde and retrograde membrane traffic between the ER and Golgi is only just beginning to be understood. Any model to explain this system, however, must account for the morphologic characteristics of the membranes involved. Whereas anterograde traffic involves discontinuous "coated" structures moving from peripheral sites in the ER toward the central Golgi, retrograde traffic utilizes continuous "noncoated" tubule structures that move from a central site (i.e., the CGN) to the peripheral ER (see Figure 3). Such a system maximizes volume transport (utilizing vacuolar structures) in the anterograde direction and membrane transport (utilizing tubules) in the retrograde direction. It is therefore ideal for sorting of bulk flow lumenal contents from recycling membrane early in the biosynthetic pathway.

Animals↗

PubMed1996

The mammalian Golgi apparatus during M-phase.

The Golgi apparatus in mammalian cells disassembles into several thousand vesicles as cells enter M-phase. Disassembly is dependent on the action of cdc2-kinase and at least two pathways contribute to the fragmentation: One involves the budding of COP-coated vesicles from Golgi cisternae with concomitant inhibition of fusion with their target membranes, the other is a less well characterised COP-independent pathway. During telophase, the Golgi fragments reassemble and fuse into a fully functional Golgi stack, using at least two distinct fusion pathways. The morphological changes of the Golgi apparatus during M-phase offer an ideal system to study how cellular organelles are generated and how their structure is maintained during interphase.

Animals↗

PubMed1984

Effect of monensin on the Golgi apparatus of absorptive cells in the small intestine of the rat. Morphological and cytochemical studies.

The effect of short-time treatment with the ionophore monensin, administered intraluminally at concentrations of 5 and 10 microM, 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 cis- or 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.

Acid Phosphatase↗