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1.
Rat liver and canine pancreas rough endoplasmic reticulum-derived vesicles, which were sealed and of the same topographical orientation as in vivo, were used in a system in vitro to demonstrate translocation of ATP into their lumen. Translocation of ATP is saturable (apparent Km: 3-4 microM and Vmax: 3-7 pmol/min/mg of protein) and protein mediated because treatment of intact vesicles with Pronase, N-ethylmaleimide, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibit transport. The entire ATP molecule is being translocated; this was shown by high performance liquid chromatography analysis and the use of a nonhydrolyzable analog. Control experiments rule out that translocation of ATP attributed to rough endoplasmic reticulum-derived vesicles is due to contamination by mitochondria and Golgi vesicles. Following translocation of ATP into the lumen of the vesicles, binding to luminal proteins including BiP (immunoglobulin heavy chain-binding protein-glucose-regulated protein 78) and glucose-regulated protein 94 was observed. This binding appeared to be specific because similar experiments with GTP were negative. These studies strongly suggest that translocation of ATP into the rough endoplasmic reticulum lumen may serve as a mechanism for making ATP available in proposed energy requiring reactions within the lumen.  相似文献   

2.
A Golgi vesicle preparation isolated from the mammary tissue of rats in mid-lactation has been shown to contain the caseins of rat milk. These proteins were phosphorylated when the Golgi vesicles were incubated in the presence of [gamma-32P]ATP. Although this phosphorylation occurred when the physical integrity of the vesicles was maintained, it was markedly increased when the membrane structure was disrupted by hypoosmotic conditions or by use of detergents. The kinase responsible has been shown to be responsive to the intravesicular concentration of Ca2+ and to the extravesicular concentration of Mg2+. These results have been interpreted in terms of a model suggesting a transmembrane location for the enzyme with binding sites on the cytosolic membrane face for Mg2+ and possibly also for ATP and on the luminal surface for Ca2+ and the caseins. Others have postulated that the assembly of caseins into micelles occurs in Golgi vesicles and requires both prior phosphorylation of the proteins and the presence of Ca2+. In this investigation we demonstrate that treatments which increase the intravesicular casein phosphorylation also alter the Ca2+ balance within the vesicle lumen. These results are discussed in relation to the ATP-dependent accumulation of Ca2+ by the mammary gland Golgi vesicles.  相似文献   

3.
Anion channels provide a pathway for Cl influx into the lumen of the Golgi cisternae. This influx permits luminal acidification by the organelle's H+-ATPase. Three different experimental approaches, electrophysiological, biochemical, and proteomic, demonstrated that two Golgi anion channels, GOLAC-1 and GOLAC-2, also mediate ATP anion transport into the Golgi lumen. First, GOLAC-1 and -2 were incorporated into planar lipid bilayers, and single-channel recordings were obtained. Low ionic activities of K2ATP added to the cis-chamber directly inhibited the Cl subconductance levels of both channels, with Km values ranging from 16 to 115 µM. Substitution of either K2ATP or MgATP for Cl on the cis, trans, or both sides indicated that ATP is conducted by the channels with a relative permeability sequence of Cl > ATP4– > MgATP2–. Single-channel currents were observed at physiological concentrations of Cl and ATP, providing evidence for their importance in vivo. Second, transport of [-32P]ATP into sealed Golgi vesicles that maintain in situ orientation was consistent with movement through the GOLACs because it exhibited little temperature dependence and was saturated with an apparent Km = 25 µM. Finally, after transport of [-32P]ATP, a protease-protection assay demonstrated that proteins are phosphorylated within the Golgi lumen, and after SDS-PAGE, the proteins in the phosphorylated bands were identified by mass spectrometry. GOLAC conductances, [-32P]ATP transport, and protein phosphorylation have identical pharmacological profiles. We conclude that the GOLACs play dual roles in the Golgi complex, providing pathways for Cl and ATP influx into the Golgi lumen. Golgi complex; Cl channel; mass spectrometry; phosphorylation  相似文献   

4.
Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.  相似文献   

5.
Previous studies in vitro on proteoglycan biosynthesis from our laboratory have shown that nucleotide sugar precursors of all the sugars of the linkage oligosaccharides (xylose, galactose, and glucuronic acid) and of the glycosaminoglycans (N-acetylglucosamine, N-galactosamine, and glucuronic acid) are transported by specific carriers into the lumen of Golgi vesicles. More recently, we also reported the reconstitution in phosphatidylcholine liposomes of detergent-solubilized Golgi membrane proteins containing transport activities of CMP-sialic acid and adenosine-3'-phosphate-5'-phosphosulfate. We have now completed the successful reconstitution into liposomes of the Golgi membrane transport activities of UDP-galactose, UDP-xylose, and UDP-glucuronic acid. Transport of these nucleotide sugars into Golgi protein proteoliposomes occurred with the same affinity, temperature dependence, and sensitivity to inhibitors as observed with intact Golgi vesicles. Preloading of proteoliposomes with UMP, the putative antiporter for Golgi vesicle transport of these nucleotide sugars, stimulated transport of the nucleotide sugars by 2-3-fold. Transport of UDP-xylose into Golgi protein proteoliposomes was dependent on the presence of endogenous Golgi membrane lipids while that of UDP-galactose and UDP-glucuronic acid was not. This suggests a possible stabilizing or regulatory role for Golgi lipids on the UDP-xylose translocator. Finally, we have also shown that detergent-solubilized Golgi membrane translocator proteins can be partially purified by an ion-exchange chromatographic step before successful reconstitution into liposomes, demonstrating that this reconstitution approach can be used for the biochemical purification of these transporters.  相似文献   

6.
We have studied in rat liver the subcellular sites and topography of xylosylation and galactosylation reactions occurring in the biosynthesis of the D-glucuronic acid-galactose-galactose-D-xylose linkage region of proteoglycans and of glucuronosylation reactions involved in both glycosaminoglycan biosynthesis and bile acid and bilirubin conjugation. The specific translocation rate of UDP-xylose into sealed, "right-side-out" vesicles from the Golgi apparatus was 2-5-fold higher than into sealed right-side-out vesicles from the rough endoplasmic reticulum (RER). Using the above vesicle preparations, we only detected endogenous acceptors for xylosylation in the Golgi apparatus-rich fraction. The specific activity of xylosyltransferase (using silk fibroin as exogenous acceptor) was 50-100-fold higher in Golgi apparatus membranes than in those from the RER. Previous studies had shown that UDP-galactose is translocated solely into vesicles from the Golgi apparatus. In these studies, we found the specific activity of galactosyltransferase I to be 40-140-fold higher in membranes from the Golgi apparatus than in those from the RER. The specific translocation rate of UDP-D-glucuronic acid into vesicles from the Golgi apparatus was 10-fold higher than into those from the RER, whereas the specific activity of glucuronosyltransferase (using chondroitin nonasaccharide as exogenous acceptor) was 12-30-fold higher in Golgi apparatus membranes than in those from the RER. Together, the above results strongly suggest that, in rat liver, the biosynthesis of the above-described proteoglycan linkage region occurs in the Golgi apparatus. The specific activity of glucuronosyltransferase, using bile acids and bilirubin as exogenous acceptor, was 10-25-fold higher in RER membranes than those from the Golgi apparatus. This suggests that transport of UDP-D-glucuronic acid into the RER lumen is not required for such reactions.  相似文献   

7.
Glycosylation, sulfation and phosphorylation of proteins, proteoglycans and lipids occur in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must be first transported from the cytosol into the Golgi lumen by specific transporters. The topology and structure of these hydrophobic, multi-transmembrane-spanning proteins are beginning to be understood.  相似文献   

8.
《The Journal of cell biology》1995,131(6):1377-1386
Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro- alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.  相似文献   

9.
The transfer of precursor proteins through the membrane of the rough endoplasmic reticulum (ER) in yeast is strictly dependent on the presence of ATP. Since Kar2p (the yeast homologue of mammalian BiP) is required for translocation, and is an ATP binding protein, an ATP transport system must be coupled to the translocation machinery of the ER. We report here the characterization of a transport system for ATP in vesicles derived from yeast ER. ATP uptake into vesicles was found to be saturable in the micromolar range with a Km of 1 x 10(-5) M. ATP transport into ER vesicles was specifically inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a stilbene derivative known to inhibit a number of other anion transporters, and by 3'-O-(4-benzoyl)benzoyl-ATP (Bz2-ATP). Inhibition of ATP uptake into yeast microsomes by DIDS and Bz2-ATP blocked protein translocation in vitro measured co- as well as post-translationally. The inhibitory effect of DIDS on translocation was prevented by coincubation with ATP. Moreover, selective membrane permeabilization, allowing ATP access to the lumen, restored translocation activity to DIDS-treated membranes. These results demonstrate that translocation requires a DIDS and Bz2-ATP-sensitive component whose function is to transport ATP to the lumen of the ER. These findings are consistent with current models of protein translocation in yeast which stipulate the participation of Kar2p in the translocation process.  相似文献   

10.
It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.  相似文献   

11.
Madin-Darby canine kidney cells are more resistant than most other cell types to the classical effects of brefeldin A (BFA) treatment, the induction of retrograde transport of Golgi cisternae components to the endoplasmic reticulum. Here we show that sulfation of heparan sulfate proteoglycans (HSPGs), chondroitin sulfate proteoglycans (CSPGs), and proteins in the Golgi apparatus is dramatically reduced by low concentrations of BFA in which Golgi morphology is unaffected and secretion still takes place. BFA treatment seems to reduce sulfation by inhibition of the uptake of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) into the Golgi lumen, and the inhibitory effect of BFA was similar for HSPGs, CSPGs, and proteins. This was different from the effect of chlorate, a well known inhibitor of PAPS synthesis in the cytoplasm. Low concentrations of chlorate (2-5 mm) inhibited sulfation of CSPGs and proteins only, whereas higher concentrations (15-30 mm) were required to inhibit sulfation of HSPGs. Golgi fractions pretreated with BFA had a reduced capacity for the synthesis of glycosaminoglycans (GAGs), but control level capacity could be restored by the addition of cytosol from various sources. This indicates that the PAPS pathway to the Golgi lumen depends on a BFA-sensitive factor that is present both on Golgi membranes and in the cytoplasm.  相似文献   

12.
When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.  相似文献   

13.
Entamoeba histolytica is a protozoan parasite that causes dysentery in developing countries of Africa, Asia, and Latin America. The lack of a defined Golgi apparatus in E. histolytica as well as in other protists led to the hypothesis that they had evolved prior to the acquisition of such organelle even though glycoproteins, glycolipids, and antigens have been detected, the latter of which react with antibodies against Golgi apparatus proteins of higher eukaryotes. We here provide direct evidence for Golgi apparatus-like functions in E. histolytica as well as for components of glycoprotein folding quality control. Using a combination of bioinformatic, cell biological, and biochemical approaches we have (a) cloned and expressed the E. histolytica UDP-galactose transporter in Saccharomyces cerevisiae; its K(m) for UDP-galactose is 2.9 microm; (b) characterized vesicles in an extract of the above protist, which transport UDP-galactose into their lumen with a K(m) of 2.7 microm;(c) detected galactosyltransferase activity(ies) in the lumen of the above vesicles with the K(m) for UDP-galactose, using endogenous acceptors, being 93 microm;(d) measured latent apyrase activities in the above vesicles, suggesting they are in the lumen; (e) characterized UDP-glucose transport activities in Golgi apparatus and endoplasmic reticulum-like vesicles with K(m)s for UDP-glucose of approximately 2-4 microm. Although the endoplasmic reticulum-like fraction showed UDP-glucose: glycoprotein glucosyltransferase activity, the Golgi apparatus-like fraction did not. This fraction contained other glucosyltransferases. Together, these studies demonstrate that E. histolytica has different vesicles that play a role in protein glycosylation and folding quality control, analogous to the above organellar functions of higher eukaryotes.  相似文献   

14.
Phosphoproteins and protein kinases of the Golgi apparatus membrane   总被引:5,自引:0,他引:5  
Incubation of a highly purified fraction derived from rat liver Golgi apparatus with [gamma-32P]ATP results in phosphorylation of several endogenous phosphoproteins. One phosphoprotein with an apparent Mr of 48,300 is radiolabeled to an apparent extent at least 5-fold higher than any other phosphoprotein as part of either the Golgi apparatus or highly purified rat liver fractions derived from the rough endoplasmic reticulum, mitochondria, plasma membrane, coated vesicles, cytosol, and total homogenate. Approximately 70% of the 48.3-kDa phosphoprotein appears to be a specific extrinsic Golgi membrane protein with the phosphorylated amino acid being threonine. The protein kinase which phosphorylates the 48.3-kDa protein is an intrinsic Golgi membrane protein and is dependent on Mg2+, independent of Ca2+, calmodulin, and cAMP, and is inhibited by N-ethylmaleimide. Preliminary evidence suggests that there are also intrinsic membrane protein kinases in the Golgi apparatus which are dependent on Ca2+ and cAMP. The physiological role of the above phosphoproteins and protein kinases is not known.  相似文献   

15.
We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.  相似文献   

16.
ABSTRACT: BACKGROUND: In yeast and mammals, many plasma membrane (PM) proteins destined for degradation are tagged with ubiquitin. These ubiquitinated proteins are internalized into clathrin-coated vesicles and are transported to early endosomal compartments. There, ubiquitinated proteins are sorted by the endosomal sorting complex required for transport (ESCRT) machinery into the intraluminal vesicles of multivesicular endosomes. Degradation of these proteins occurs after endosomes fuse with lysosomes/lytic vacuoles to release their content into the lumen. In plants, some PM proteins, which cycle between the PM and endosomal compartments, have been found to be ubiquitinated, but it is unclear whether ubiquitin is sufficient to mediate internalization and thus acts as a primary sorting signal for the endocytic pathway. To test whether plants use ubiquitin as a signal for the degradation of membrane proteins, we have translationally fused ubiquitin to different fluorescent reporters for the plasma membrane and analyzed their transport. RESULTS: Ubiquitin-tagged PM reporters localized to endosomes and to the lumen of the lytic vacuole in tobacco mesophyll protoplasts and in tobacco epidermal cells. The internalization of these reporters was significantly reduced if clathrin-mediated endocytosis was inhibited by the coexpression of a mutant of the clathrin heavy chain, the clathrin hub. Surprisingly, a ubiquitin-tagged reporter for the Golgi was also transported into the lumen of the vacuole. Vacuolar delivery of the reporters was abolished upon inhibition of the ESCRT machinery, indicating that the vacuolar delivery of these reporters occurs via the endocytic transport route. CONCLUSIONS: Ubiquitin acts as a sorting signal at different compartments in the endomembrane system to target membrane proteins into the vacuolar degradation pathway: If displayed at the PM, ubiquitin triggers internalization of PM reporters into the endocytic transport route, but it also mediates vacuolar delivery if displayed at the Golgi. In both cases, ubiquitin-tagged proteins travel via early endosomes and multivesicular bodies to the lytic vacuole. This suggests that vacuolar degradation of ubiquitinated proteins is not restricted to PM proteins but might also facilitate the turnover of membrane proteins in the early secretory pathway.  相似文献   

17.
A Driouich  G F Zhang    L A Staehelin 《Plant physiology》1993,101(4):1363-1373
Brefeldin A (BFA), a specific inhibitor of Golgi-mediated secretion in animal cells, has been used to study the organization of the secretory pathway and the function of the Golgi apparatus in plant cells. To this end, we have employed a combination of electron microscopical, immunocytochemical, and biochemical techniques to investigate the effects of this drug on the architecture of the Golgi apparatus as well as on the secretion of proteins and complex cell wall polysaccharides in sycamore maple (Acer pseudoplatanus) suspension-cultured cells. We have used 2.5 and 7.5 micrograms/mL of BFA, which is comparable to the 1 to 10 micrograms/mL used in experiments with animal cells. Electron micrographs of high-pressure frozen and freeze-substituted cells show that although BFA causes swelling of the endoplasmic reticulum cisternae, unlike in animal cells, it does not induce the disassembly of sycamore maple Golgi stacks. Instead, BFA induces the formation of large clusters of Golgi stacks, an increase in the number of trans-like Golgi cisternae, and the accumulation in the cytoplasm of very dense vesicles that appear to be derived from trans Golgi cisternae. These vesicles contain large amounts of xyloglucan (XG), the major hemicellulosic cell wall polysaccharide, as shown by immunocytochemical labeling with anti-XG antibodies. All of these structural changes disappear within 120 min after removal of the drug. In vivo labeling experiments using [3H]leucine demonstrate that protein secretion into the culture medium, but not protein synthesis, is inhibited by approximately 80% in the presence of BFA. In contrast, the incorporation of [3H]fucose into N-linked glycoproteins, which occurs in trans-Golgi cisternae, appears to be affected to a greater extent than the incorporation of [3H]xylose, which has been localized to medial Golgi cisternae. BFA also affects secretion of complex polysaccharides as evidenced by the approximate 50% drop in incorporation of [3H]xylose and [3H]fucose into cell wall hemicelluloses. Taken together, these findings suggest that at concentrations of 2.5 to 7.5 mu g/mL BFA causes the following major changes in the secretory pathway of sycamore maple cells: (a) it inhibits the transport of secretory proteins to the cell surface by about 80% and of hemicelluloses by about 50%; (b) it changes the patterns of glycosylation of N-linked glycoproteins and hemicelluloses; (c) it reduces traffic between trans Golgi cisternae and secretory vesicles; (d) it produces a major block in the transport of XG-containing, dense secretory vesicles to the cell surface; and (e) it induces the formation of large aggregates of Golgi apparatus of plant and animal cels share many functional and structural characteristics, the plant Golgi apparatus possesses properties that make its response to BFA unique.  相似文献   

18.
Copper is essential for a variety of important biological processes as a cofactor and regulator of many enzymes. Incorporation of copper into the secreted and plasma membrane-targeted cuproenzymes takes place in Golgi, a compartment central for normal copper homeostasis. The Golgi complex harbors copper-transporting ATPases, ATP7A and ATP7B that transfer copper from the cytosol into Golgi lumen for incorporation into copper-dependent enzymes. The Golgi complex also sends these ATPases to appropriate post-Golgi destinations to ensure correct Cu fluxes in the body and to avoid potentially toxic copper accumulation. Mutations in ATP7A or ATP7B or in the proteins that regulate their trafficking affect their exit from Golgi or subsequent retrieval to this organelle. This, in turn, disrupts the homeostatic Cu balance, resulting in copper deficiency (Menkes disease) or copper overload (Wilson disease). Research over the last decade has yielded significant insights into the enzymatic properties and cell biology of the copper ATPases. However, the mechanisms through which the Golgi regulates trafficking of ATP7A/7B and, therefore, maintains Cu homeostasis remain unclear. This review summarizes current data on the role of the Golgi in Cu metabolism and outlines questions and challenges that should be addressed to understand ATP7A and ATP7B trafficking mechanisms in health and disease.  相似文献   

19.
In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.  相似文献   

20.
Procollagen (PC)-I aggregates transit through the Golgi complex without leaving the lumen of Golgi cisternae. Based on this evidence, we have proposed that PC-I is transported across the Golgi stacks by the cisternal maturation process. However, most secretory cargoes are small, freely diffusing proteins, thus raising the issue whether they move by a transport mechanism different than that used by PC-I. To address this question we have developed procedures to compare the transport of a small protein, the G protein of the vesicular stomatitis virus (VSVG), with that of the much larger PC-I aggregates in the same cell. Transport was followed using a combination of video and EM, providing high resolution in time and space. Our results reveal that PC-I aggregates and VSVG move synchronously through the Golgi at indistinguishable rapid rates. Additionally, not only PC-I aggregates (as confirmed by ultrarapid cryofixation), but also VSVG, can traverse the stack without leaving the cisternal lumen and without entering Golgi vesicles in functionally relevant amounts. Our findings indicate that a common mechanism independent of anterograde dissociative carriers is responsible for the traffic of small and large secretory cargo across the Golgi stack.  相似文献   

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