Golgi apparatus function in membrane transformations and product compartmentalization: studies with cell fractions isolated from rat liver.
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Protein kinase C bound to the Golgi apparatus supports the formation of constitutive transport vesicles.
Constitutive secretion of heparan sulphate proteoglycans (HSPGs) was stimulated in human hepatoma HepG2 cells by phorbol 12-myristate 13-acetate (PMA) and inhibited by calphostin C, a specific inhibitor of protein kinase C (PKC). To delineate more closely the site of PKC action, the packaging in vitro of 35SO4-labelled HSPGs into transport vesicles was investigated. Formation of transport vesicles at the trans-Golgi network was stimulated by PMA and inhibited by calphostin C or Ro 31-8220 by using a post-nuclear supernatant. Treatment of either isolated Golgi-enriched membranes or cytosolic proteins with calphostin C provided evidence that membrane-bound PKC forms strongly supported vesicle formation, whereas cytosolic PKC forms showed a marginal effect. The PKC isoforms PKC-alpha and PKC-zeta were attached to highly purified Golgi membranes, as shown by Western blotting. Both isoforms were localized by confocal immunofluorescence microscopy in the Golgi area of HepG2 cells. Immunoelectron microscopy of ultrathin cryosections of HepG2 cells showed that PKC-zeta predominantly attaches to the trans-Golgi region, whereas PKC-alpha binds to the cis- and trans-Golgi area.
Carcinoma, Hepatocellular↗
On the involvement of the Golgi apparatus and GERL in processing of secretion products and some enzyme activities in the rat pituitary mammotroph.
Whole pituitary of Wistar albino rats were either (a) immediately fixed in an aldehyde fixative (Karnovsky), or incubated prior to the fixation in (b) unaltered medium 199, (c) medium 199 supplemented with 1.65 M sucrose, or (d) medium 199 diluted with distilled water (1:2). Incubation time was for 0.5 h, at a temperature of 4 degrees or 37 degrees C. All specimens were routinely processed for acid phosphatase (AcPase) or thiamine pyrophosphatase (TPPase) demonstration and examined ultrastructurally. In the control series a and b AcPase activity in the mammotroph was associated with the cisternae of GERL, dense bodies, coated vesicles, immature secretion granules often continuous with cisternae of GERL, and occasionally with the innermost lamellae of the of the Goldi apparatus (GA). TPPase activity was found within the innermost lamellae and the fenestrated element of GA. Occasional granular cores were seen within TPPase-positive lamellae. In all mammotrophs of series (4 degrees and 37 degrees C) severe shrinkage of the lamellae of GA and other membrane-bound structures in the Golgi area was apparent. In some mammotrophs of the series c (37 degrees C) AcPase activity, in addition to its usual sites, was revealed within all lamellae of GA, as well as in some tubular and fenestrated elements continuous with GA. The innermost AcPase-positive lamellae of GA were sometimes partially separated from the others and continuous with immature secretion granules or dense bodies, thus qualifying as cisternae of GERL in the course of their gradual formation from the Golgi lamellae. A gradual increase in AcPase activity from the cis to trans poles of GA could sometimes be noted. TPPase activity in this series spread over ll lamellae of GA and its fenestrated elements. The occasional profiles of rough endoplasmic reticulum and some smooth-surfaced vesicles near GA also gave positive reactions for the enzyme. At 4 degrees C the distributions of both enzyme activities were as in the controls. In all series d (4 or 37 degrees C) the membrane-bounded organelles of the mammotroph were greatly swollen except for occasional cells (at 37 degrees C) in which all lamellae of GA were paradoxically shrunk as in series c. The distributions of AcPase and TPPase activities in series d were as in the controls, except for some cells with shrunken lamellae (at 37 degrees C) in which TPPase activity was spread throughout GA. The results suggest that experimental dehydration of GA in the mammotroph acts by overcoming latency of AcPase and TPPase or by accelerating maturation of AcPase and TPPase activities from their hypothetical precursors thereby tracing the intracellular pathways of these enzymes. It also follows that GA may participate in the initial stage of secretion-granule formation and enzyme activity maturation whereas GERL finalizes the process.
Acid Phosphatase↗
Zebrafish fat-free is required for intestinal lipid absorption and Golgi apparatus structure.
The zebrafish fat-free (ffr) mutation was identified in a physiological screen for genes that regulate lipid metabolism. ffr mutant larvae are morphologically indistinguishable from wild-type sibling larvae, but their absorption of fluorescent lipids is severely impaired. Through positional cloning, we have identified a causative mutation in a highly conserved and ubiquitously expressed gene within the ffr locus. The Ffr protein contains a Dor-1 like domain typical of oligomeric Golgi complex (COG) gene, cog8. Golgi complex ultrastructure is disrupted in the ffr digestive tract. Consistent with a possible role in COG-mediated Golgi function, wild-type Ffr-GFP and COG8-mRFP fusion proteins partially colocalize in zebrafish blastomeres. Enterocyte retention of an endosomal lipid marker in ffr larvae support the idea that altered vesicle trafficking contributes to the ffr mutant defect. These data indicate that ffr is required for both Golgi structure and vesicular trafficking, and ultimately lipid transport.
Amino Acid Sequence↗
Possible role of the Golgi apparatus in the assembly of very low density lipoprotein.
Transit of newly synthesized triacyl[3H]-glycerol through organelles of the secretory system leading to assembly into nascent very low density lipoproteins (VLDLs) or to cytoplasmic storage was investigated in chick hepatocytes. Cells in monolayer culture were pulse-labeled with [2-3H]glycerol, and after different periods of chase with unlabeled glycerol, the movement of triacyl[3H]glycerol through the endoplasmic reticulum (ER) and Golgi and the incorporation into nascent VLDL and cytoplasmic triacylglycerol-rich vesicles (TGRVs) were determined. Initially, newly synthesized triacyl[3H]glycerol is tightly associated with the ER. Movement from the ER of triacyl[3H]glycerol destined for cytoplasmic storage (as TGRVs) is extremely rapid and virtually complete within 8 min of chase. After 8 min of chase, triacyl[3H]glycerol lost from organelles of the secretory system was accounted for entirely as triacyl[3H]glycerol secreted as VLDL. Comparison of the rates of movement of triacyl[3H]glycerol, apolipoprotein B, apolipoprotein II, and apolipoprotein A-I through the ER and Golgi and of their secretion in nascent VLDL suggests that assembly of triacyglycerol with apolipoproteins occurs in the Golgi. Experiments with permeabilized hepatocytes supplemented with cytosol show that newly synthesized triacyl[3H]glycerol and [3H]phospholipid moves from the ER through the full-density range of Golgi elements and is dependent upon supplementary ATP.
Animals↗
Protein retention and localization in the endoplasmic reticulum and the golgi apparatus.
Protein transport along the secretory pathway is supported by a noria of vesicles that bud and fuse, load and unload their cargo from one compartment into the other. However, despite this constant flow-through of proteins and lipids the various compartments of the secretory pathway are able to maintain their own specific composition. Here, we discuss recent insights into mechanisms of protein retention and localization that are necessary for the maintenance of endoplasmic reticulum (ER)- and Golgi-associated typical functions such as protein folding and glycosylation in plant cells.
Animals↗
A 19-kDa protein belonging to a new family is expressed in the Golgi apparatus of neural cells.
The mouse 8.5 mRNA encodes a 171-residue novel protein which displays a highly significant similarity with the product of the previously characterized neuronal p1A75 cDNA (Sutcliffe, J.G., Milner, R.J., Shinnick, T.M., and Bloom, F.E. (1983) Cell 33, 671-682). Northern blot and in situ hybridization experiments indicated that the 8.5 mRNA is specifically expressed in neural and neuroendocrine tissues. An affinity-purified antibody directed against the recombinant 8.5 protein demonstrated the existence of the 19-kDa natural protein in brain and evidenced its prominent juxtanuclear Golgi-like localization in cultured neurons. Ultrastructural analysis of the same preparation revealed a specific labeling of all the Golgi saccules and of some vesicles in the Golgi zone. In transfected COS cells, the exogenous protein was also detected in the Golgi area, indicating, therefore, the presence of a Golgi targeting signal in its primary sequence.
Amino Acid Sequence↗
A region containing a proline-rich motif targets sG(i2) to the golgi apparatus.
The central function of heterotrimeric GTP-binding proteins (G proteins) is the transduction of extracellular signals, via membrane receptors, leading to the activation of intracellular effectors. In addition to being associated with the plasma membrane, the alpha subunits of some of these proteins have also been localized in intracellular compartments. The mRNA of the G-protein inhibitory alpha subunit 2 (G(alphai2)) encodes two proteins, G(alphai2) and sG(i2), by an alternative splicing mechanism. sG(i2) differs from G(alphai2) in the C-terminal region and localizes in the Golgi in contrast to the plasma membrane localization of G(alphai2). In this paper we show that the sequence specific to sG(i2) can direct the Golgi localization of other G(alphai) subunits, but not of the stimulatory subunit G(alphas) or of a secreted protein. This indicates that, in addition to the sG(i2) C-terminus, sequences located elsewhere in the protein are required to determine the Golgi localization. Inside the sG(i2) C-terminal region we have identified a 14-amino-acid proline-rich motif which specifies the Golgi localization. Finally, we show that the sG(i2) subunit, once activated, leaves the Golgi to be localized in the endoplasmic reticulum.
Amino Acid Sequence↗
Yeast rab GTPase-activating protein Gyp1p localizes to the Golgi apparatus and is a negative regulator of Ypt1p.
A family of related proteins in yeast Saccharomyces cerevisiae is known to have in vitro GTPase-activating protein activity on the Rab GTPases. However, their in vivo function remains obscure. One of them, Gyp1p, acts on Sec4p, Ypt1p, Ypt7p, and Ypt51p in vitro. Here, we present data to reveal its in vivo substrate and the role that it plays in the function of the Rab GTPase. Red fluorescent protein-tagged Gyp1p is concentrated on cytoplasmic punctate structures that largely colocalize with a cis-Golgi marker. Subcellular fractionation of a yeast lysate confirmed that Gyp1p is peripherally associated with membranes and that it cofractionates with Golgi markers. This localization suggests that Gyp1p may only act on Rab GTPases on the Golgi. A gyp1Delta strain displays a growth defect on synthetic medium at 37 degrees C. Overexpression of Ypt1p, but not other Rab GTPases, strongly inhibits the growth of gyp1Delta cells. Conversely, a partial loss-of-function allele of YPT1, ypt1-2, can suppress the growth defect of gyp1Delta cells. Furthermore, deletion of GYP1 can partially suppress growth defects associated with mutants in subunits of transport protein particle complex, a complex that catalyzes nucleotide exchange on Ypt1p. These results establish that Gyp1p functions on the Golgi as a negative regulator of Ypt1p.
Carboxypeptidases↗
Fine structure of the cell surface and Golgi apparatus of Ochromonas.
Ochromonas danica has an unusually flexible cell surface capable of producing projections of varying sizes and shapes: large projections, 340-360 nm long, and small projections, 50-110 nm long. These projections have been demonstrated by transmission and scanning electron microscopy; some of them may break off into the medium and be the source of extracellular membranes and vesicles reported in the cell-free O. danica growth medium. Ruthernium red stained the acid mucopolysaccharide layer just outside the cell surface as well as small blebs at the cell surface. The Golgi complex of O. danica, Ochromonas malhamensis, Ochromonas sociabilis and Ochromonas sp. produced small coated vesicles which may move toward and fuse with the plasma membrane. The role of the several vesicles is unknown but possible functions are discussed.
Animals↗
Foot-and-mouth disease virus replication sites form next to the nucleus and close to the Golgi apparatus, but exclude marker proteins associated with host membrane compartments.
Picornavirus infection of cells generally results in the production of membranous vesicles containing the viral proteins necessary for viral RNA synthesis. To determine whether foot-and-mouth disease virus (FMDV) infection induced similar structures, and which cellular components were involved, the subcellular distribution of FMDV proteins was compared with protein markers of cellular membrane compartments. Using immunofluorescence analysis and digital deconvolution, it was shown that FMDV structural and non-structural proteins co-localize to punctate structures in juxtanuclear virus assembly sites close to the Golgi complex. Significantly, viral protein 2C did not co-localize with marker proteins of the cis- or medial-Golgi compartments or trans-Golgi network. Furthermore, incubation of infected cells with brefeldin A caused a redistribution of Golgi proteins to the endoplasmic reticulum, but did not affect the distribution of 2C and, by inference, the integrity of the virus assembly site. Taken with the observation that 2C was membrane-associated, but failed to fractionate with Golgi markers on density gradients, it was possible to conclude that Golgi membranes were not a source of structures containing 2C. Further immunofluorescence analysis showed that 2C was also separate from marker proteins of the endoplasmic reticulum, endoplasmic reticulum intermediate compartment, endosomes and lysosomes. The results suggest that the membranes generated at FMDV assembly sites are able to exclude organelle-specific marker proteins, or that FMDV uses an alternative source of membranes as a platform for assembly and replication.
Animals↗
PLD1b in IIC9 fibroblasts is selectively activated in the nucleus and not in the Golgi apparatus.
Mitogen-induced activation of a nuclear-acting PC-phospholipase D (PLD) is mediated, at least in part, by the translocation of RhoA to the nucleus. A remaining question is whether PLD in all subcellular compartments is regulated in the same manner. To address this question, we identified PLD in another subcellular compartment and determined whether its activity was influenced by alpha-thrombin in a RhoA-dependent manner. The data in this manuscript show that nuclear PLD is selectively regulated. alpha-Thrombin stimulates an increase in PLD activity in IIC9 fibroblast nuclei while Golgi PLD activity is unaffected. We cloned PLD1 from IIC9s (hamPLD1b) to show that it is present in both nuclei and Golgi. Interestingly, only nuclear PLD1 is modulated by alpha-thrombin, demonstrating that this activity is selectively regulated. These data provide support for the physiological importance of agonist-induced nuclear signalling enzymes.
ADP-Ribosylation Factors↗
Human cyclins B1 and B2 are localized to strikingly different structures: B1 to microtubules, B2 primarily to the Golgi apparatus.
We have raised and characterized antibodies specific for human cyclin B2 and have compared the properties of cyclins B1 and B2 in human tissue culture cells. Cyclin B1 and B2 levels are very low in G1 phase, increase in S and G2 phases and peak at mitosis. Both B-type cyclins associate with p34cdc2; their associated kinase activities appear when cells enter mitosis and disappear as the cyclins are destroyed in anaphase. However, human cyclins B1 and B2 differ dramatically in their subcellular localization. Cyclin B1 co-localizes with microtubules, whereas cyclin B2 is primarily associated with the Golgi region. In contrast to cyclin B1, cyclin B2 does not relocate to the nucleus at prophase, but becomes uniformly distributed throughout the cell. The different subcellular locations of human cyclins B1 and B2 implicate them in the reorganization of different aspects of the cellular architecture at mitosis and indicate that different mitotic cyclin-cyclin-dependent kinase complexes may have distinct roles in the cell cycle.
Amino Acid Sequence↗
Localization of myocilin to the golgi apparatus in Schlemm's canal cells.
PURPOSE: Biochemical and genetic evidence suggests that overexpression of or mutations in myocilin within the cells of the aqueous humor outflow pathway play a significant role in the development of steroid-induced and several other open-angle glaucomas. As a baseline to understanding the normal and pathologic function of myocilin, we determined the subcellular localization of myocilin in steroid-treated human Schlemm's canal endothelial (SC) cells. METHODS: SC cells were grown to confluence, treated with dexamethasone for 10 days, and then stained using antibodies against myocilin, tubulin, or beta-COP (a specific golgi protein) or vital stains for endoplasmic reticulum (ER) and golgi. Brefeldin A (BFA) and nocodazol (NZ) were used to disrupt the golgi or microtubules. RESULTS: The authors found that myocilin staining was (a) always centered around the centrosome, (b) very similar to the pattern seen with NBD-ceramide, (c) was disrupted in characteristic ways by BFA and NZ and (d) showed extensive colocalization with beta-COP. CONCLUSIONS: Results indicate that myocilin is localized to the golgi in SC cells. Such localization is consistent with myocilin being processed for secretion but is also consistent with sequence analysis and other data that suggest that myocilin or myocilin mutations might be targeted to the cytoplasmic face of the golgi, and under some circumstances play a role in or interfere with golgi or vesicle function. How such interference could eventually lead to open angle glaucoma is discussed.
Brefeldin A↗
Cytosolic phospholipase A2-alpha and cyclooxygenase-2 localize to intracellular membranes of EA.hy.926 endothelial cells that are distinct from the endoplasmic reticulum and the Golgi apparatus.
Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme that plays an important role in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid can be converted subsequently into prostacyclin, a potent vasodilator and inhibitor of platelet activation, through the action of cyclooxygenase (COX) enzymes. Here we study the relocation of cPLA2-alpha in human EA.hy.926 endothelial cells following stimulation with the calcium-mobilizing agonist, A23187. Relocation of cPLA2-alpha was seen to be highly cell specific, and in EA.hy.926 cells occurred primarily to intracellular structures resembling the endoplasmic reticulum (ER) and Golgi. In addition, relocation to both the inner and outer surfaces of the nuclear membrane was observed. Colocalization studies with markers for these subcellular organelles, however, showed colocalization of cPLA2-alpha with nuclear membrane markers but not with ER or Golgi markers, suggesting that the relocation of cPLA2-alpha occurs to sites that are separate from these organelles. Colocalization with annexin V was also observed at the nuclear envelope, however, little overlap with staining patterns for the potential cPLA2-alpha interacting proteins, annexin I, vimentin, p11 or actin, was seen in this cell type. In contrast, cPLA2-alpha was seen to partially colocalize specifically with the COX-2 isoform at the ER-resembling structures, but not with COX-1. These studies suggest that cPLA2-alpha and COX-2 may function together at a distinct and novel compartment for eicosanoid signalling.
Annexin A1↗
Ricin-binding properties of acid hydrolases from isolated lysosomes implies prior processing by terminal transferases of the trans-Golgi apparatus.
Acid hydrolases were isolated from the lysosome fraction of beta-galactosidase-deficient human fibroblasts and from the mannose 6-phosphate containing medium in which they were grown. Nearly half of the total beta-hexosaminidase and beta-glucuronidase from both sources bound to Ricin specifically. Lysosomal beta-hexosaminidase, metabolically labelled with [35S]-methionine, was also fractionated on Ricin-agarose. SDS-PAGE of immunoprecipitates from Ricin-binding and non-binding fractions revealed approximately equivalent amounts of cross-reacting material at the appropriate MW. We interpret these results to mean that acid hydrolases which are segregated to lysosomes are exposed to trans-Golgi processing enzymes to about the same extent as enzymes which are secreted, and that segregation by the Man 6-P receptor occurs after transit through the trans-Golgi compartment.
Binding Sites↗
SNAREs and membrane fusion in the Golgi apparatus.
Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell. They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs. Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion. NSF is an ATPase which will disrupt complexes composed of different SNAREs. However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane. The essential role of NSF may be to prime SNAREs for a direct role during fusion. The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi. The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar. Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes. Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles. In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue. There are only eight members of the syntaxin family in yeast. Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex. They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi. We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles.
Animals↗
A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells.
Protein secretion plays an important role in plant cells as it does in animal and yeast cells, but the tools to study molecular events of plant secretion are very limited. We have focused on the Sar1 GTPase, which is essential for the vesicle formation from the endoplasmic reticulum (ER) in yeast, and have previously shown that tobacco and Arabidopsis SAR1 complement yeast sar1 mutants. In this study, we have established a transient expression system of GFP-fusion proteins in tobacco and Arabidopsis cultured cells. By utilizing confocal laser scanning microscopy, we demonstrate that a dominant negative mutant of Arabidopsis Sar1 inhibits the ER-to-Golgi transport of Golgi membrane proteins, AtErd2 and AtRer1B, and locates them to the ER. The same mutant Sar1 also blocks the exit from the ER of a vacuolar storage protein, sporamin. These results not only provide the first evidence that the Sar1 GTPase functions in the ER-to-Golgi transport in plant cells, but also prove that conditional expression of dominant mutants of secretory machinery can be a useful tool in manipulating vesicular trafficking.
Arabidopsis↗