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• W2024263598 abstract "We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that α-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of α-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of β-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on β-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far. We have investigated the effect of nordihydroguaiaretic acid (NDGA), an inhibitor of lipoxygenase, on the intracellular protein transport and the structure of the Golgi complex. Pulse-chase experiments and immunoelectron microscopy showed that NDGA strongly inhibits the transport of newly synthesized secretory proteins to the Golgi complex resulting in their accumulation in the endoplasmic reticulum (ER). Despite their retention in the ER, oligosaccharides of secretory and ER-resident proteins were processed to endoglycosidase H-resistant forms, raising the possibility that oligosaccharide-processing enzymes are redistributed from the Golgi to the ER. Morphological observations further revealed that α-mannosidase II (a cis/medial-Golgi marker), but not TGN38 (a trans-Golgi network marker), rapidly redistributes to the ER in the presence of NDGA, resulting in the disappearance of the characteristic Golgi structure. Upon removal of the drug, the Golgi complex was reassembled into the normal structure as judged by perinuclear staining of α-mannosidase II and by restoration of the secretion. These effects of NDGA are quite similar to those of brefeldin A. However, unlike brefeldin A, NDGA did not cause a dissociation of β-coatomer protein, a subunit of coatomer, from the Golgi membrane. On the contrary, NDGA exerted the stabilizing effect on β-coatomer protein/membrane interaction against the dissociation caused by brefeldin A and ATP depletion. Taken together, these results indicate that NDGA is a potent agent disrupting the structure and function of the Golgi complex with a mechanism different from those known for other drugs reported so far. Newly synthesized secretory proteins are transported from the endoplasmic reticulum (ER) 1The abbreviations used are: ER, endoplasmic reticulum; ARF, ADP-ribosylation factor; BFA, brefeldin A; C3, third component of complement; CGN, cis-Golgi network; COP, coatomer protein complex; DPP IV, dipeptidyl-peptidase IV; endo H, endo-β-hexosaminidase H; Man II, α-mannosidase II; NDGA, nordihydroguaiaretic acid; α1-PI, α1-protease inhibitor; TGN, trans-Golgi network; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-3-O-(thio)triphosphate. to the cell surface via the Golgi complex, which is comprised of structurally and functionally distinct subcompartments including the cis-Golgi network, the Golgi stack, and the trans-Golgi network (TGN) (1Rothman J.E. Orci L. Nature. 1992; 335: 409-415Crossref Scopus (744) Google Scholar). In the secretory pathway, the Golgi complex plays a key role in the sorting and modification of proteins. Resident ER proteins are sorted at the cis-Golgi network and recycled back to the ER (2Letouneur F. Gaynor E.C. Hennecke S. Demouliere C. Duden R. Emer S. Riezmen H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (674) Google Scholar), and lysosomal proteins are sorted at the TGN (3Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (767) Google Scholar). Modifications such as oligosaccharide processing (4Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar), proteolytic processing (5Misumi Y. Oda K. Fujiwara T. Takami N. Tashiro K. Ikehara Y. J. Biol. Chem. 1991; 266: 16954-16959Abstract Full Text PDF PubMed Google Scholar), and sulfation (6Hirose S. Oda K. Ikehara Y. J. Biol. Chem. 1988; 263: 7426-7430Abstract Full Text PDF PubMed Google Scholar) are carried out by enzymes with specific localizations in the Golgi subcompartments. Each transport step along the secretory pathway is mediated by small vesicles that bud from a donor compartment and fuse with a target compartment membrane. Biochemical and genetic studies have identified a number of proteins involved in the vesicular transport (1Rothman J.E. Orci L. Nature. 1992; 335: 409-415Crossref Scopus (744) Google Scholar, 7Waters M.G. Serafini T. Rothman J.E. Nature. 1991; 349: 248-251Crossref PubMed Scopus (381) Google Scholar, 8Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromonas S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 9Salama N.R. Schekman R.W. Curr. Opin. Cell Biol. 1995; 7: 536-543Crossref PubMed Scopus (66) Google Scholar). These include coat protein complexes (COPI and COPII) and ADP-ribosylation factor (ARF) or Sar1p for vesicle budding, and N-ethylmaleimide-sensitive fusion protein, solubleN-ethylmaleimide-sensitive fusion attachment proteins, and soluble N-ethylmaleimide-sensitive fusion attachment protein receptors (SNAREs) for vesicle docking and fusion. It is postulated that the specificity of vesicle targeting is generated by complexes formed between membrane proteins on the transport vesicles (v-SNAREs) and those on the target compartments (t-SNAREs) (10Rothman J.E. Nature. 1994; 372: 55-63Crossref PubMed Scopus (2011) Google Scholar). The use of drugs affecting the secretory process at distinct sites in the cell may prove valuable for more detailed studies of specific steps in secretion and may lead to the understanding of the molecular basis of the mechanisms involved in intracellular transport. One of the most useful and characterized drugs is the fungal metabolite brefeldin A (BFA). BFA strongly blocks secretion by apparently inhibiting protein transport from the ER to the Golgi (11Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar, 12Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar) and causes redistribution of resident Golgi proteins into the ER (12Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar, 13Fujiwara T. Oda K. Ikehara Y. Cell Struct. Funct. 1989; 14: 605-616Crossref PubMed Scopus (35) Google Scholar, 14Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1315) Google Scholar, 15Doms R.W. Russ G. Yewdel J.W. J. Cell Biol. 1989; 109: 61-72Crossref PubMed Scopus (429) Google Scholar). The primary action of the drug is now believed to be to inhibit Golgi membrane-catalyzed GDP/GTP exchange of ARF (16Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (596) Google Scholar, 17Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (585) Google Scholar) that is required for assembly of ARF and COPI onto the Golgi membrane (18Orci L. Palmer D.J. Amherdt M. Rothman J.E. Nature. 1993; 364: 732-734Crossref PubMed Scopus (182) Google Scholar), resulting in lack of formation of transport vesicles. The redistribution of resident Golgi proteins to the ER in the presence of BFA is microtubule-dependent (19Lippincott-Schwartz J. Donaldson J.G. Schweizer A. Berger E.G. Hauri H.-P. Yuan L.C. Klausner R.D. Cell. 1990; 60: 821-836Abstract Full Text PDF PubMed Scopus (736) Google Scholar) and suggests the existence of a retrograde transport pathway without involving COPI vesicles. This is in contrast to the recent evidence that resident ER proteins are retrieved from the cis-Golgi network by COPI-dependent transport vesicles (2Letouneur F. Gaynor E.C. Hennecke S. Demouliere C. Duden R. Emer S. Riezmen H. Cosson P. Cell. 1994; 79: 1199-1207Abstract Full Text PDF PubMed Scopus (674) Google Scholar), which also raises a question as to how COPI proteins are involved in both the anterograde and retrograde transport from the cis-Golgi network (9Salama N.R. Schekman R.W. Curr. Opin. Cell Biol. 1995; 7: 536-543Crossref PubMed Scopus (66) Google Scholar, 20Pelham H.R.B. Cell. 1994; 79: 1125-1127Abstract Full Text PDF PubMed Scopus (107) Google Scholar). Thus, more data are required to elucidate the details of the vesicular transport mechanism. Nordihydroguaiaretic acid (NDGA), a polyhydroxyphenolic antioxidant, is known to exert the inhibitory effect on lipoxygenase pathways of arachidonic acid metabolism (21Morris H.R. Piper P.J. Taylor G.W. Tippins J.R. Br. J. Pharmacol. 1979; 66: 452Google Scholar, 22Armour C.L. Hughes J.M. Seale J.P. Temple D.M. Eur. J. Biochem. 1981; 72: 93-96Google Scholar). It was recently demonstrated that NDGA also inhibits the secretion of prolactin from GH3 cells (23Tagaya M. Henomatsu N. Yoshimori T. Yamamoto A. Tashiro Y. Fukui T. FEBS Lett. 1993; 324: 210-214Crossref Scopus (35) Google Scholar) and the intracellular transport of vesicular stomatitis virus G protein (24Tagaya M. Henomatsu N. Yoshimori T. Yamamoto A. Tashiro Y. Mizushima S. J. Biochem. (Tokyo). 1996; 119: 863-869Crossref PubMed Scopus (22) Google Scholar). The drug is likely to inhibit protein transport from the ER to the Golgi and also from the TGN to the cell surface. In the present study we examined the effect of NDGA on secretion and intracellular processing of secretory proteins, confirming that the drug indeed blocks the protein transport from the ER to the Golgi complex. In addition, we have found that NDGA rapidly disrupts the cisternal organization of the Golgi complex and causes the redistribution of resident Golgi proteins into the ER without dissociation of COPI from the membrane in contrast to the effects of BFA. NDGA was obtained from Sigma. BFA was from Wako Chemicals (Osaka, Japan). Antibodies to β-COP (25Seguchi T. Goto Y. Ono M. Fujiwara T. Shimada T. Kung H. Nishioka M. Ikehara Y. Kuwano M. J. Biol. Chem. 1992; 267: 11626-11630Abstract Full Text PDF PubMed Google Scholar) and dipeptidyl-peptidase IV (DPP IV) (26Ogata S. Misumi Y. Ikehara Y. J. Biol. Chem. 1989; 264: 3596-3601Abstract Full Text PDF PubMed Google Scholar) were raised in rabbits as described. Anti-TGN38 antibody was generously provided by Dr. G. Banting (University of Bristol, U.K.) and monoclonal anti-α-mannosidase II (Man II) was from Berkeley Antibody Co. (Richmond, CA). Antibodies to albumin, third component of complement (C3), and α1-protease inhibitor (α1-PI) were purchased from Organon Teknika (Durham, NC). Fluorescein isothiocyanate-conjugated anti-rabbit IgG and tetramethylrhodamine isothiocyanate-conjugated anti-mouse IgG were from Dakopatts (Glostrup, Denmark). Peroxidase-conjugated goat anti-rabbit IgG was from Biosys (Compiegne, France). Biotinylated anti-mouse IgG and peroxidase-conjugated streptavidin were from Vector Laboratories (Burlingame, CA). HepG2 and H35P15 cells were cultured in Eagle's minimum essential medium containing 10% fetal calf serum. NRK and COS-1 cells were cultured in Dulbecco's modified Eagle's minimum essential medium with 10% fetal calf serum. Transfection experiments were carried out as described (27Tsuji E. Misumi Y. Fujiwara T. Takami N. Ogata S. Ikehara Y. Biochemistry. 1992; 31: 11921-11927Crossref PubMed Scopus (52) Google Scholar, 28Fujiwara T. Tsuji E. Misumi Y. Takami N. Ikehara Y. Biochem. Biophys. Res. Commun. 1992; 185: 776-784Crossref PubMed Scopus (17) Google Scholar), for which a plasmid encoding a mutant DPP IV (mDPP IV) with substitution of Gly633 → Arg was constructed. The plasmid (20 μg) was transfected into COS-1 cells with the Lipofectin Reagent. The transfected cells were cultured for 2 days before use. Cells in 60-mm plastic dishes were pulse-labeled for 10 or 20 min with [35S]methionine (50 μCi/dish) in methionine-free Eagle's minimum essential medium and chased in the complete Eagle's minimum essential medium in the presence or absence of NDGA (30 μm) or BFA (5 μg/ml) (11Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar). Cycloheximide (50 μm) was added to the chase medium. At the indicated times, the cells were separated from the medium, and cell lysates were prepared (29Takami N. Ogata S. Oda K. Misumi Y. Ikehara Y. J. Biol. Chem. 1988; 263: 3016-3021Abstract Full Text PDF PubMed Google Scholar). 35S-Labeled proteins of cell lysates and medium were immunoprecipitated with the indicated antibodies, extensively washed, and subjected to enzyme digestion when indicated (29Takami N. Ogata S. Oda K. Misumi Y. Ikehara Y. J. Biol. Chem. 1988; 263: 3016-3021Abstract Full Text PDF PubMed Google Scholar). Samples were incubated with endo H (0.2 unit/ml) in 50 mm acetate buffer (pH 5.5) at 37 °C for 16 h, or with neuraminidase (0.2 unit/ml) in 50 mmacetate buffer (pH 5.0) for 20 h. Proalbumin (pI 6.0) and albumin (pI 5.8) were separated by electrofocusing on 5% polyacrylamide gels (pH range from 5 to 8) as described previously (11Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar). SDS-PAGE was carried out on 7.5% gels (for C3) and on 10% gels (for α1-PI) according to Laemmli (30Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and the gels were processed for fluorography (29Takami N. Ogata S. Oda K. Misumi Y. Ikehara Y. J. Biol. Chem. 1988; 263: 3016-3021Abstract Full Text PDF PubMed Google Scholar). HepG2 cells (8 × 106 cells/150-mm dish), before or after the NDGA treatment, were washed and suspended in phosphate-buffered saline (PBS). An equal volume of 0.6 m perchloric acid was added to the cell suspension and mixed well, followed by centrifugation at 32,000 ×g for 5 min. The resulting supernatant was neutralized with 5 m KOH and used for the determination of ATP as described (11Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar). NRK cells were suspended in 0.2 ml of a buffer containing 25 mm Hepes-KOH (pH 7.2), 115 mm KCl, 2.5 mm magnesium acetate, 1 mm dithiothreitol and 0.2 m sucrose (buffer A) and homogenized by passing 15 times through a 25-gauge needle. Nuclei and cell debris were removed by centrifugation at 600 ×g for 5 min. The postnuclear supernatant was separated into cytosol and membrane fractions by centrifugation at 125,000 ×g for 1 h. Proteins of each sample were separated by SDS-PAGE (8% gels) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with rabbit anti-β-COP IgG (25 μg/ml) for 1 h, followed by incubation with peroxidase-conjugated anti-rabbit IgG for 1 h. The immunoreaction was visualized by an enhanced chemiluminescence detection system (Sigma). Cells were grown on glass coverslips. After the indicated treatments, cells were briefly washed with PBS and fixed with 3% paraformaldehyde in PBS for 15 min at room temperature. The fixed cells were washed, permeabilized with 0.1% saponin in PBS and incubated with the indicated primary antibodies for 15 min, followed by incubation for 15 min with fluorescein isothiocyanate- or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies. To see the membrane association of β-COP, cells on a coverslip were perforated according to Simons and Vitra (31Simons K. Vitra H. EMBO J. 1987; 8: 2241-2247Crossref Scopus (68) Google Scholar) and washed before fixation and immunostaining. Briefly, a HATF filter (0.45-μm pore size, Millipore) presoaked in buffer A was placed on a cell monolayer for 1 min. The filter was gently peeled off the cell monolayer on the coverslip, and the perforated cells were washed with buffer A for 5 min at room temperature. Drugs, when indicated, were present throughout the perforation and washing steps. The cells were then washed with PBS, fixed, and stained for β-COP as above. HepG2 cells (for albumin) and H35P15 cells (for Man II) were fixed for 2 h with the paraformaldehyde/lysine/periodate fixative (32McLean I.W. Nakane P.K. J. Histochem. Cytochem. 1974; 22: 1077-1083Crossref PubMed Scopus (3200) Google Scholar) and permeabilized with 0.05% saponin (12Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar). The HepG2 cells were incubated for 2 h with anti-albumin antibodies, followed by incubation for 1 h with peroxidase-conjugated goat anti-rabbit IgG. The H35P15 cells were incubated with anti-Man II antibodies and then with biotinylated anti-mouse IgG and peroxidase-conjugated streptavidin for 1 h each. After the peroxidase reaction, the cells were processed for electron microscopy as described previously (12Fujiwara T. Oda K. Yokota S. Takatsuki A. Ikehara Y. J. Biol. Chem. 1988; 263: 18545-18552Abstract Full Text PDF PubMed Google Scholar). HepG2 cells synthesize various plasma proteins including albumin, the C3, and α1-PI. In addition, albumin and C3 are initially synthesized as proforms, which are converted into mature forms at the TGN (5Misumi Y. Oda K. Fujiwara T. Takami N. Tashiro K. Ikehara Y. J. Biol. Chem. 1991; 266: 16954-16959Abstract Full Text PDF PubMed Google Scholar, 11Misumi Y. Misumi Y. Miki K. Takatsuki A. Tamura G. Ikehara Y. J. Biol. Chem. 1986; 261: 11398-11403Abstract Full Text PDF PubMed Google Scholar). In control cells, newly synthesized albumin was rapidly converted to the mature form and secreted into the medium (Fig. 1 A). The presence of a small amount of proalbumin in the medium may be due to the relatively low activity of the converting enzyme in HepG2 cells as compared with that of hepatocytes (33Oda K. Fujiwara T. Ikehara Y. Biochem. J. 1990; 265: 161-167Crossref PubMed Scopus (26) Google Scholar). In the presence of 30 μm NDGA, however, the processing and secretion of albumin were strongly blocked (Fig. 1 B). Upon removal of the drug, the labeled albumin was normally processed and secreted into the medium (Fig. 1 C), indicating the reversibility of the drug effect. Since protein synthesis was found to be significantly inhibited at higher concentrations of the drug, we used NDGA at 30 μmthroughout the following experiments. Essentially the same results were obtained for the processing and secretion of pro-C3. In the control cells, pro-C3 synthesized as a single polypeptide of 180 kDa and was processed into the α (115 kDa) and β (65 kDa) subunits, which were secreted into the medium, although a considerable amount of the proform was also secreted (Fig. 1 D). The processing and secretion of pro-C3 were completely blocked by NDGA (Fig. 1 E), and this inhibitory effect was reversible (Fig. 1 F). These results suggest that NDGA blocks secretion by inhibiting the intracellular transport of proteins before the site where the proforms of albumin and C3 are processed into the mature forms. Immunofluorescence microscopy showed that albumin was markedly concentrated in the juxtanuclear region in the control cells (Fig. 2 A), whereas the protein was distributed in reticular network structures extending throughout the cytoplasm in the NDGA-treated cells (Fig. 2 B). The alteration in localization of albumin was examined in more detail by immunoelectron microscopy. In the control cells, albumin was most heavily stained in the Golgi complex, although also detectable in the ER and nuclear envelope (Fig. 2 C). In the cells treated with the drug for 2 h (Fig. 2 D), however, we could not identify the characteristic Golgi stack structure where albumin had been concentrated. The immunoreaction product was detected exclusively in the ER and nuclear envelope. Although some mitochondria appeared to be slightly swollen, structures of other organelles were not significantly changed by treatment with the drug. These results suggest that NDGA primarily blocks the protein transport from the ER, resulting in the accumulation of secretory proteins in the ER. Since it is known that intracellular protein transport is critically dependent on cellular ATP levels, we examined the effect of NDGA on the ATP level in HepG2 cells. The ATP content in control cells was measured to be about 17 nmol/mg protein. When cells were treated with the drug, the cellular ATP level was rapidly decreased to about 40% of the control, a level that was maintained throughout an incubation time of up to 3 h with the drug (Fig. 3). However, removal of the drug from the medium at 30 min of incubation did not allow the ATP level to be recovered during the following incubation times without the drug. This is in contrast to the reversible effect of NDGA on secretion and proteolytic processing (Fig. 1). These results suggest that the inhibitory effect of NDGA on protein transport may not be directly coupled with the reduction in the cellular ATP levels caused by the drug. If NDGA blocks the transport of secretory glycoproteins out of the ER, it is expected that their oligosaccharides will remain as high mannose type sensitive to endo H digestion. To test this, we examined the biosynthesis and processing of the secretory glycoprotein α1-PI in HepG2 cells. In control cells, α1-PI was initially synthesized as a 51-kDa form and subsequently converted to a 56-kDa form, which was secreted into the medium (Fig. 4 A, lanes 1–6). The 51-kDa form was sensitive to endo H digestion, whereas the 56-kDa form was resistant to endo H (Fig. 4 A, lanes 7–11). In addition, the latter form decreased the molecular size to 51 kDa when treated with neuraminidase (Fig. 4 A, lane 12). In contrast, in NDGA-treated cells, the newly synthesized 51-kDa form was neither converted into the mature 56-kDa form nor secreted into the medium (Fig. 4 B, lanes 1–6). The 51-kDa form in the treated cells, however, showed different responses to endo H depending on the chase times. Upon digestion with endo H, the protein was initially converted to the completely sensitive 41-kDa form, then to partially sensitive forms, and finally to a single resistant 51-kDa form (Fig. 4 B, lanes 7–11). The final 51-kDa form obtained at 3-h chase was insensitive to neuraminidase treatment (Fig. 4 B, lane 13) and had the same molecular size as that of the neuraminidase-treated form in the control cells (Fig. 4 A, lane 12). The results indicate that the 51-kDa form contains complex type oligosaccharides without sialylation. Immunofluorescence microscopy confirmed that α1-PI was retained in the ER-like structures in the treated cells (Fig. 4 D). The finding that the oligosaccharides of α1-PI retained in the ER are converted to the complex type might suggest that the processing enzymes localized in the Golgi complex are redistributed into the ER in the presence of NDGA. This possibility was confirmed by another set of experiments. DPP IV is an ectoenzyme withN-linked oligosaccharide chains (26Ogata S. Misumi Y. Ikehara Y. J. Biol. Chem. 1989; 264: 3596-3601Abstract Full Text PDF PubMed Google Scholar). We found that a mutant with substitution of Gly633 → Arg (mDPP IV) is retained in the ER and degraded there without being transported to the Golgi complex (27Tsuji E. Misumi Y. Fujiwara T. Takami N. Ogata S. Ikehara Y. Biochemistry. 1992; 31: 11921-11927Crossref PubMed Scopus (52) Google Scholar, 28Fujiwara T. Tsuji E. Misumi Y. Takami N. Ikehara Y. Biochem. Biophys. Res. Commun. 1992; 185: 776-784Crossref PubMed Scopus (17) Google Scholar). When mDPP IV was expressed in COS-1 cells by transfection, the protein was retained in the ER (Fig. 5 D) and remained completely sensitive to endo H even after 4-h chase in control cells (Fig. 5 A). In the presence of NDGA, however, the protein acquired the resistance to endo H after the chase (Fig. 5 B) as observed in BFA-treated cells (Fig. 5 C). There was no significant difference in distribution of mDPP IV between the control and treated cells, demonstrating its retention in the ER (Fig. 5, D and E). These results suggest that NDGA causes the redistribution of the Golgi-resident processing enzymes to the ER as effectively as BFA. Man II has been used as a cis/medial-Golgi marker (4Kornfeld R. Kornfeld S. Annu. Rev. Biochem. 1985; 54: 631-664Crossref PubMed Scopus (3779) Google Scholar,14Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1315) Google Scholar), whereas TGN38 is a membrane protein localized in the TGN (34Luzio J.P. Burke B. Banting G. Howell K.E. Braghetta P. Stanley K.K. Biochem. J. 1990; 270: 97-102Crossref PubMed Scopus (258) Google Scholar). Changes in localizations of Man II and TGN38 were examined in NRK cells as a function of time after exposure to NDGA. Man II localized in perinuclear regions in control cells (Fig. 6 A) still remained in the same regions with slightly fragmented and dispersed structures in cells treated with the drug for 5 min (Fig. 6 C). After 30 min of incubation, Man II was distributed on punctate or reticular structures over the cytoplasm, and no significant perinuclear staining was detected (Fig. 6 E). A more intense staining pattern for Man II on the reticular structures was observed in cells exposed for 2 h (Fig. 6 G). This effect of NDGA was found to be reversible. The reticular distribution of Man II in cells treated with the drug for 1 h (Fig. 7 A) completely reversed to a perinuclear localization after 2 h of incubation without the drug (Fig. 7 D) through intermediate stepwise changes (Fig. 7, B and C) reciprocal to those observed in the presence of the drug (Fig. 6, C and E). In contrast to Man II, the localization of TGN38 was not so significantly changed by the drug (Fig. 6, B, D, F, and H), indicating that NDGA has little effect, if any, on the TGN structure.Figure 7Reversibility of the NDGA effect on the localization of Man II. NRK cells were pretreated with NDGA for 1 h at 37 °C. The cells were then incubated in a fresh medium without the drug for 0 min (A), 30 min (B), 1 h (C), and 2 h (D), fixed, permeabilized, and stained for Man II by immunofluorescence as in Fig. 6. Bar = 10 μm.View Large Image Figure ViewerDownload (PPT) The change in localization of Man II was examined by immunoelectron microscopy, for which rat hepatoma H35P15 cells were used since Man II was more intensely stained in these cells than in the NRK cells. Immunofluorescence microscopy confirmed the same staining profiles of Man II in H35P15 cells (Fig. 8, A and B) as in NRK cells (Fig. 6, A and G). When observed by immunoelectron microscopy, Man II was detected only in the Golgi cisternae in control cells (Fig. 8 C). In contrast, no immunostainable Golgi structures could be observed in cells treated with NDGA for 3 h. Instead, Man II was clearly detected in the ER and weakly detected in the nuclear envelope (Fig. 8 D). Taken together, these results indicate that the cis/medial-Golgi protein, but not the TGN marker, redistribute into the ER in the presence of NDGA. The data presented here show that the effects of NDGA are quite similar to those caused by BFA. BFA is known to prevent the attachment of ARF and coatomer (COPI) to the Golgi membrane, resulting in their rapid dissociation from the membrane (16Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (596) Google Scholar, 17Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (585) Google Scholar, 35Donaldson J.G. Lippincott-Schwartz J. Klausner R.D. J. Cell Biol. 1990; 111: 2295-2306Crossref PubMed Scopus (292) Google Scholar). To see whether NDGA also has the same effect, we examined the response of β-COP, a subunit of COPI, upon NDGA treatment. β-COP was colocalized with Man II in the Golgi complex in control cells (Fig. 9, panel A, a and b). In cells treated with NDGA for 10 min, β-COP was detected in large vesicular structures scattered in the cytoplasm and colocalized with Man II (Fig. 9, panel A, c and d). After 30 min of incubation, β-COP and Man II were dispersed into the cytoplasm and showed essentially the same staining profile (Fig. 9, panel A, e and f). Immunoelectron microscopy confirmed that β-COP was localized to the ER and nuclear envelope in the treated cells (data not shown) as observed for Man II (Fig. 8 D). When cells were perforated and washed, β-COP was completely removed from cells that had been treated with BFA (Fig. 9, panel B, c and d), whereas the dispersed β-COP remained in the NDGA-treated cells (Fig. 9, panel B, a and b). In addition, an immunoblot" @default.
• W2024263598 created "2016-06-24" @default.
• W2024263598 creator A5016460971 @default.
• W2024263598 creator A5020968941 @default.
• W2024263598 creator A5056261551 @default.
• W2024263598 creator A5060033416 @default.
• W2024263598 date "1998-01-01" @default.
• W2024263598 modified "2023-09-30" @default.
• W2024263598 title "Nordihydroguaiaretic Acid Blocks Protein Transport in the Secretory Pathway Causing Redistribution of Golgi Proteins into the Endoplasmic Reticulum" @default.
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