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• W2009698855 abstract "Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply. Adaptation of eukaryotic cells to changing environmental conditions entails rapid regulation of protein targeting and transport to specific organelles. Such adaptation is well exemplified in mammalian cells exposed to nitrogen starvation that are triggered to form and transport autophagosomes to lysosomes, thus constituting an inducible intracellular trafficking pathway. Here we investigated the relationship between the general secretory machinery and the autophagic pathway in Chinese hamster ovary cells grown in the absence of amino acid. Utilizing VSVG-YFP (vesicular stomatitis virus G protein fused to yellow fluorescent protein) and norepinephrine as markers for constitutive and regulated exocytosis, respectively, we found that secretion is attenuated in cells grown in media lacking amino acid. Such decrease in exocytosis stems from partial inhibition of N-ethylmaleimide-sensitive factor ATPase activity, which in turn causes an accumulation of SNARE complexes at both the Golgi apparatus and the plasma membrane of the starved cells. These findings expose a novel cellular strategy to attenuate secretion of proteins under conditions of limited amino acid supply. Specific recognition between an intracellular vesicle carrying cargo molecules and its appropriate target membrane involves the interaction between v-SNAREs, 1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; SNAP, soluble NSF attachment protein; v-SNARE, vesicle-associated SNARE; t-SNARE, target membrane SNARE; VSVG, vesicular stomatitis virus G protein; YFP, yellow fluorescent protein; NSF, N-ethylmaleimide-sensitive factor; GATE-16, Golgi-associated ATPase enhancer, 16 kDa; PM, plasma membrane; NE, norepinephrine; NEM, N-ethylmaleimide; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; FCS, fetal calf serum; DMEM, Dulbecco's modified Eagle's medium; α-MEM, minimum essential medium; PIPES, 1,4-piperazinediethanesulfonic acid. integral membrane proteins located on the vesicle, and t-SNAREs, located at the target membrane (1Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1310) Google Scholar). These interactions form a SNARE “core complex,” which consists of four entwined α-helix bundles of typically three Q-SNARE helices and one R-SNARE helix, a classification based on conserved glutamine or arginine residues at the center of their SNARE-binding domain. The SNARE core complex is stabilized mainly by hydrophobic interactions between the four helices and by a central ionic layer consisting of one arginine and three glutamine residues contributed by each of the four α-helices (2Sutton R.B. Fasshauer D. Jahn R. Brunger A.T. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1929) Google Scholar). Formation of transcomplexes of SNAREs from opposing membranes yields a close, stable proximity between the two membranes, which facilitates overcoming the energy barrier required for membrane fusion (3Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2019) Google Scholar, 4Parlati F. Weber T. McNew J.A. Westermann B. Söllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (217) Google Scholar, 5Nickel W. Weber T. McNew J.A. Parlati F. Söllner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12571-12576Crossref PubMed Scopus (160) Google Scholar). Furthermore, using three sets of functionally identified yeast t-SNAREs to mediate the fusion of ER-derived transport vesicles with the Golgi, the homotypic fusion of vacuoles, and the fusion with the plasma membrane (PM), it was demonstrated that isolated SNARE proteins encode compartmental specificity and mediate the actual fusion event (6McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (533) Google Scholar, 7Parlati F. McNew J.A. Fukuda R. Miller R. Sollner T.H. Rothman J.E. Nature. 2000; 407: 194-198Crossref PubMed Scopus (209) Google Scholar, 8Parlati F. Varlamov O. Paz K. McNew J.A. Hurtado D. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5424-5429Crossref PubMed Scopus (147) Google Scholar). Additional proteins are required for targeting and tethering of these transport vesicles with their specific targets (9Waters M.G. Hughson F.M. Traffic. 2000; 1: 588-597Crossref PubMed Scopus (91) Google Scholar). The hexameric ATPase N-ethylmaleimide-sensitive factor (NSF) utilizes ATP hydrolysis to dissociate cis-SNARE complexes after membrane fusion, allowing the individual SNARE proteins to be recycled for subsequent rounds of fusion (10Mayer A. Wickner W. Haas A. Cell. 1996; 85: 83-94Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar, 11Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1584) Google Scholar). Whereas specific v- and t-SNAREs are associated with each intercompartmental transport step, NSF is a general cytosolic factor that can disassemble SNARE complexes from most intracellular transport steps. The ATPase activity of NSF is enhanced by α-SNAP (soluble NSF attachment protein), which mediates NSF binding to different SNARE complexes (12Whiteheart S.W. Schraw T. Matveeva E.A. Int. Rev. Cytol. 2001; 207: 71-112Crossref PubMed Scopus (101) Google Scholar), and possibly by other factors such as GATE-16 (13Muller J.M. Shorter J. Newman R. Deinhardt K. Sagiv Y. Elazar Z. Warren G. Shima D.T. J. Cell Biol. 2002; 157: 1161-1173Crossref PubMed Scopus (70) Google Scholar, 14Sagiv Y. Legesse-Miller A. Porat A. Elazar Z. EMBO J. 2000; 19: 1494-1504Crossref PubMed Scopus (209) Google Scholar) and rab-6 (15Han S.Y. Park D.Y. Park S.D. Hong S.H. Biochem. J. 2000; 352: 165-173Crossref PubMed Scopus (23) Google Scholar). Notably, evidence for direct negative regulation of NSF activity in vivo has been suggested by Matsushita et al. (16Matsushita K. Morrell C.N. Cambien B. Yang S.X. Yamakuchi M. Bao C. Hara M.R. Quick R.A. Cao W. O'Rourke B. Lowenstein J.M. Pevsner J. Wagner D.D. Lowenstein C.J. Cell. 2003; 115: 139-150Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar), whereby S-nitrosylation of specific cysteine residues on NSF leads to attenuation of triggered exocytosis of endothelial granules. However, it is not known yet whether NSF S-nitrosylation controls other cellular membrane fusion processes such as constitutive exocytosis or vesicular transport along the secretory pathway. Direct regulation of vesicular trafficking is crucial not only for the spatially and temporally controlled secretion of bioactive molecules but also for controlling the levels of various molecules (e.g. receptors, transporters) on the PM (17Grusovin J. Macaulay S.L. Front. Biosci. 2003; 8: 620-641Crossref PubMed Google Scholar). A variety of regulatory events that operate at different steps of vesicular transport control the trafficking along specific cellular pathways in response to different signals. Developmental, environmental, and cell cycle-related signals have differential effects on different vesicular transport pathways. For example, transport along the secretory pathway in Xenopus oocytes is blocked between the trans-Golgi and the PM during meiotic maturation (18Leaf D.S. Roberts S.J. Gerhart J.C. Moore H.P. Dev. Biol. 1990; 141: 1-12Crossref PubMed Scopus (36) Google Scholar). On the other hand, transport of special vesicles to lysosomes is enhanced upon amino acid deprivation in a process known as autophagy (19Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (764) Google Scholar). Autophagy is a bulk protein degradation process in which newly formed double membrane vesicles, termed autophagosomes, deliver cytoplasmic contents and organelles for lysosomal degradation (19Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (764) Google Scholar). There are at least three types of autophagy: macroautophagy (referred to as autophagy hereafter), microautophagy (20Kunz J.B. Schwarz H. Mayer A. J. Biol. Chem. 2004; 279: 9987-9996Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), and chaperon-mediated autophagy (21Cuervo A.M. Gomes A.V. Barnes J.A. Dice J.F. J. Biol. Chem. 2000; 275: 33329-33335Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Autophagy, a relatively understudied membrane-trafficking process, may be regarded as a unique vesicular transport pathway that is triggered in response to stress conditions such as nitrogen starvation (19Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (764) Google Scholar). Recent studies implicated transport factors such as Sec18p (NSF yeast homologue), SNAREs, Rabs, and members of GATE-16 family in fusion steps associated with autophagy (22Abeliovich H. Dunn Jr., W.A. Kim J. Klionsky D.J. J. Cell Biol. 2000; 151: 1025-1034Crossref PubMed Scopus (236) Google Scholar, 23Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (298) Google Scholar, 24Ishihara N. Hamasaki M. Yokota S. Suzuki K. Kamada Y. Kihara A. Yoshimori T. Noda T. Ohsumi Y. Mol. Biol. Cell. 2001; 12: 3690-3702Crossref PubMed Scopus (297) Google Scholar, 25Munafo D.B. Colombo M.I. Traffic. 2002; 3: 472-482Crossref PubMed Scopus (163) Google Scholar, 26Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5503) Google Scholar). In the present study we have investigated whether deprivation of amino acid also affects other vesicular transport pathways such as constitutive and regulated secretion. By comparing exocytosis in starved versus non-starved control cells, we conclude that the Golgi to PM transport is significantly attenuated under amino acid starvation conditions. Structural and functional assays suggest that this attenuation is directly caused by inhibition of NSF ATPase activity during starvation. Our data provide, for the first time, a molecular explanation for the attenuation of exocytosis under these physiological stress conditions. Cell Culture and Induction of Amino Acid Starvation—CHO cells were grown in α-MEM containing 10% FCS, 100 units/ml penicillin, and 100 μg/ml streptomycin, either on plates or in suspension. PC12 cells were grown in DMEM containing 6% FCS, 6% horse serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. To induce autophagy by amino acid deprivation, cells were washed three times with Earl's balanced salt solution (EBSS) and then incubated in EBSS (supplemented with vitamins and pyruvate to the same concentrations present in the rich media) for the indicated time periods at 37 °C. As a control, washed cells were incubated with α-MEM or DMEM in the absence of FCS for the indicated starvation periods. Degradation and Secretion Assays—Measurement of long-lived protein degradation in CHO cells was based on an assay previously described by Ogier et al. (27Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, CHO cells plated on 6-well plates were prelabeled with [14C]valine (Amersham Biosciences) for 24 h. Cells were then washed three times with phosphate-buffered saline and then preincubated for 1 h in either α-MEM or EBSS, both containing 0.1% bovine serum albumin and 10 mm cold valine. After 1 h of incubation, the culture media were replaced with identical fresh media, and the cells were incubated for an additional 2 or 4 h. The media were collected and centrifuged for 3 min at 2200 rpm, and the supernatant was precipitated in 10% trichloroacetic acid. Total cells radioactivity was measured after lysing the cells in 0.1 m NaOH. Degradation and secretion of prelabeled proteins were measured as the ratio between the radioactivity found in the trichloroacetic acid-soluble fraction (free [14C]valine released to the medium) or in the trichloroacetic acid-insoluble fraction (incorporated [14C]valine released to the medium) and the total cells radioactivity, respectively. VSVG Trafficking—CHO cells cultured in Lab-Tek chambered coverglass system (Nunc) were transfected at 40 °C with VSVG tsO45-YFP (45Nichols B.J. Kenworthy A.K. Polishchuk R.S. Lodge R. Roberts T.H. Hirschberg K. Phair R.D. Lippincott-Schwartz J. J. Cell Biol. 2001; 153: 529-541Crossref PubMed Scopus (458) Google Scholar) by Lipofectamine according to manufacturer's protocol. After 24 h of incubation, cells were washed three times with prewarmed EBSS and further incubated at 40 °C for 3 h in control or starvation media. Cells were then shifted to 32 °C to trigger transport at t = 0. A series of fluorescent microscopy images was made at the time points indicated. To analyze the progression of VSVG along the secretory pathway under favorable versus starvation conditions, cells were scored as showing Golgi localization of VSVG when the Golgi staining intensity was at least strong as that of the ER. Cells were scored as showing PM localization of VSVG in this analysis when PM staining was clearly visualized. Representatives for each case are shown in Fig. 2. Norepinephrine Release Assay—PC12 cells were grown in poly-l-lysine-coated 6-well plate for 2–3 days until they reached 50–70% confluence. Cells were loaded at 37 °C for 16 h in DMEM containing 6% FCS, 6% horse serum and 1.5 μCi of [3H]NE (ARC Inc.)/well, rinsed with phosphate-buffered saline, and then chased in control or starvation medium for additional 3.5 h. To prevent spontaneous leakage of [3H]norepinephrine (NE) from starved cells resulting from decreased ATP cellular level, extra glucose was added to the EBSS medium to the same level found in DMEM. PC12 cells were then incubated in low K+ medium (10 mm Hepes, 5 mm KCl, 145 mm NaCl, 2 mm CaCl2, 10 mm glucose, pH 7.4) for 10 min at 37 °C prior to the induction of secretion with high K+ medium (10 mm Hepes, 55 mm KCl, 95 mm NaCl, 2 mm CaCl2, 10 mm glucose, pH 7.4). [3H]NE release was assessed in time course experiments in which the medium was collected and replaced every 2 min. Media samples were centrifuged at 3000 rpm for 3 min, and supernatant radioactivity was determined by liquid scintillation counting. At the end of the experiment, the cells were solubilized in 1 ml of 0.25 n NaOH, and the lysates were carefully collected. [3H]NE release was calculated as the ratio between the medium radioactivity and the total cells radioactivity (sum of all media counts and the radioactivity remaining in the cells at the end of the experiment). Preparation of Cytosol and Membrane Extracts—Cells were washed twice with phosphate-buffered saline and then twice in a homogenization buffer containing 0.25 m sucrose, 25 mm Tris-HCl, pH 7.4, and 50 mm KCl. The cells were then homogenized in the same buffer containing 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 2 μm pepstatin A using a Balch homogenizer (for cells grown in suspension) or Dounce homogenizer (for cells grown on plates). The homogenates were centrifuged for 5 min at 2500 rpm, and the supernatant was further centrifuged for 30 min at 200,000 × g. The supernatant containing the cytosol was collected. The pellet, containing total membranes, was resuspended in extraction buffer containing 20 mm Hepes, pH 7.0, 20 mm KCl, and 0.5% Triton X-100. To obtain cytosol and membrane extracts in the presence of ATP, CHO cells were washed twice with phosphate-buffered saline, then twice in a homogenization buffer containing 20 mm PIPES, pH 7.2, 10 mm MgCl2, 5 mm ATP, 0.5 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 0.1 m KCl, and homogenized and fractionated as described above. SNARE Complex Disassembly Assays—To compare SNARE complex levels in control versus starved cells, membrane extracts from CHO or PC12 cells were incubated in SDS-sample buffer at either 30 °C or 100 °C for 5 min and then subjected to immunoblotting. To examine the capacity of cytosolic fractions to dissociate SNARE complexes, rat brain membranes containing SNARE complexes were pretreated on ice with 1 mmN-ethylmaleimide (NEM) for 10 min followed by treatment with 2 mm dithiothreitol for 15 min. NEM-treated membranes were incubated with 50 μg of cytosol obtained from control or starved CHO cells in a reaction buffer containing 0.5 μg of α-SNAP, 0.5 mm ATP, 2 mm MgCl2, and ATP regeneration buffer at 30 °C for 30 min. In the indicated reactions, 1.2 μg of recombinant NSF was added to the disassembly assay. The membranes were then isolated by centrifugation (14,000 rpm, 10 min at 4 °C), resuspended in SDS-sample buffer, and processed for immunoblotting without prior boiling unless mentioned. Proteolytic Digestion—Recombinant NSF proteins and cytosolic fractions obtained from starved or non-starved CHO cells were incubated at 30 °C with trypsin (ratio of 1:20 and 1:200 trypsin/protein (w/w), respectively). Progress of the proteolysis was assessed by removing aliquots at the indicated time points and quenching the digestion with a 5:1 (w/w) excess of soybean trypsin inhibitor over trypsin. The aliquots were subjected to 12% SDS-PAGE and analyzed by Western blot using a mixture of 2E5, 2C8, and 6E6 anti-NSF monoclonal antibodies. Immunoprecipitation—Monoclonal antibodies (anti-NSF) and polyclonal antibodies (anti-Gos-28 or anti-syntaxin-5) were covalently coupled by dimethyl pimelimidate (Sigma) to protein G- and protein A-agarose beads (Santa Cruz Biotechnology), respectively. The coupled antibodies were then incubated with either recombinant His-NSF-Myc protein (0.5 μg) or cytosolic (200 μg) and membrane extract fractions (200 μg) obtained from control or starved cells for at least 2 h at 4 °C. Beads were washed four times in phosphate-buffered saline, the bound material was eluted by 2% SDS at 95 °C, and the eluates were subjected to Western blot analysis. In some experiments, recombinant His-NSF-Myc fractions (0.5 μg) were preincubated with cytosolic fractions (50 μg) obtained from control or starved CHO cells at 30 °C for 30 min prior to the immunoprecipitation. ATPase Activity Assay—Prior to the ATPase activity assay, NSF (6 μg) was preincubated with cytosolic fractions (40 μg) obtained from control or starved CHO cells at 30 °C for 20 min in a total volume of 100 μlof25mm Tris-HCl, pH 7.4, 50 mm KCl, 2 mm MgCl2 and 0.5 mm ATP. The assay was initiated by adding residual amounts of [γ-32P]ATP (0.25 μCi/sample), carried out for 30 min at 30 °C, and stopped by adding 100 μl of ice-cold perchloric acid to a final concentration of 10%. Nucleotides were bound to 500 μl of ice-cold charcoal, and samples were centrifuged at 14,000 rpm for 15 min at 4 °C. The resulting supernatants containing released 32Pi were quantified by liquid scintillation counting. Background activities of control and starved cytosol alone were subtracted (0.08 and 0.079 μmol of Pi/mg of protein/h for cytosolic fractions obtained from control or starved cells, respectively). Secretion Is Inhibited in Response to Amino Acid Deprivation—Autophagy triggered by starvation may be regarded as a special case of membrane-trafficking process that shares some of the components utilized by other intracellular membrane-trafficking pathways. In an attempt to study the relationship between the autophagic and the secretory pathways, we utilized [14C]valine-prelabeled tissue culture cells to examine the effects of amino acid starvation on intracellular protein degradation and simultaneously on protein secretion. In this system the autophagic activity was determined by monitoring the bulk protein degradation of long-lived proteins (27Ogier-Denis E. Houri J.J. Bauvy C. Codogno P. J. Biol. Chem. 1996; 271: 28593-28600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), whereas exocytosis was determined by monitoring the release of secreted proteins from the cells. For this purpose, prelabeled CHO cells were incubated for different time periods in either α-MEM (without FCS) or EBSS (starvation medium), and the levels of secreted labeled proteins and amino acid into the media were determined. As depicted in Fig. 1A, bulk degradation of long-lived proteins, measured by the release of trichloroacetic acidsoluble [14C]valine from the cells, was about 50% higher in cells incubated in medium lacking amino acid compared with control medium, indicating significant enhancement of autophagy in starved cells. Concomitantly, the radioactivity measured in the trichloroacetic acid-insoluble pellet was significantly reduced (∼30%) in cells deprived of amino acid (Fig. 1B). Similar results were obtained when CHO cells were prelabeled with [35S]methionine, as well as in different cell lines such as COS-7 and PC12 cells that were treated similarly (data not shown). Taken together, these results indicate that during amino acid starvation protein degradation is stimulated, whereas the constitutive secretion of newly synthesized proteins is largely inhibited. Vesicular Transport to the Plasma Membrane Is Inhibited during Starvation—To identify the step along the secretory pathway that is most affected by amino acid starvation, we utilized the well studied vesicular stomatitis virus ts045 G protein fused to yellow fluorescent protein (VSVG-YFP) to monitor trafficking through the various compartments of the secretory pathway in living cells. We took advantage of the fact that at 40 °C the ts045 VSVG mutant protein is retained within the ER, whereas upon a shift to 32 °C it moves as a synchronous population to the Golgi complex before being transported to the PM (28Presley J.F. Smith C. Hirschberg K. Miller C. Cole N.B. Zaal K.J.M. Lippincott-Schwartz J. Mol. Biol. Cell. 1998; 9: 1617-1626Crossref PubMed Scopus (73) Google Scholar). Hence, CHO cells were transfected with VSVG-YFP, incubated in normal or starvation medium for 3 h at 40 °C, and then shifted to 32 °C to trigger transport. The transport of the VSVG-YFP ts045 protein was monitored by confocal microscopy. At each of several selected time points, the percentage of cells in which the VSVG-YFP protein was localized to the ER, Golgi, or the PM was calculated. As shown in Fig. 2, the VSVG protein accumulated in the Golgi apparatus in about 60% of the control cells within a 5-min incubation period at 32 °C and in 100% of these cells within 20 min. During this period, no significant difference in the rate of transport of VSVG protein from the ER to the Golgi was detected in the starved cells. However, within 60 min the VSVG protein was detected in the PM of more than 60% of the control cells, whereas only 25% of the starved cells exhibited PM labeling. From this analysis we conclude that although ER-to-Golgi transport is only marginally affected by amino acid starvation, the transport from the Golgi apparatus to the PM is significantly attenuated in starved cells. The effect of amino acid deprivation on exocytosis was studied further in PC12 cells, a rat adrenal phaeochromocytoma cell line, which expresses several well defined neuronal properties in culture, including the regulated secretion of neurotransmitters. To examine the effects of amino acid starvation on exocytosis in this cell line, release of [3H]norepinephrine from prelabeled cells was measured in control versus starved cells. Hence, prelabeled cells were incubated for 3.5 h in either DMEM or EBSS supplemented with glucose (starvation media) and then subjected to time course secretion analysis. As depicted in Fig. 3, the basal rates of NE release were low and similar for both control and starved cells. However, when NE secretion was stimulated by directly depolarizing the PC12 cells with elevated extracellular K+ ions (55 mm KCl), 14% of the [3H]NE content was secreted within the first 2 min and cumulatively up to 31% within 12 min. Under these conditions, however, secretion of NE from the starved cells was inhibited by about 32%, indicating that triggered exocytosis too is sensitive to amino acid starvation. Disassembly of SNARE Complexes Is Inhibited during Amino Acid Starvation—A normal vesicular transport cycle requires rapid and regulated disassembly of SNARE complexes, mediated by the ATPase activity of NSF and its co-factor α-SNAP (reviewed in Ref. 29May A.P. Whiteheart S.W. Weis W.I. J. Biol. Chem. 2001; 276: 21991-21994Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Exocytosis in PC12 cells, for example, requires the formation and subsequent dissociation of a specific SNARE complex, including the vesicle-associated membrane protein (VAMP) and the PM proteins syntaxin and SNAP-25. To examine whether lack of amino acid affected the dissociation of these complexes, we relied on the fact that SNARE complexes are SDS-resistant at 37 °C and thus can be detected as high molecular weight complexes by immunoblotting (30Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar). As shown in Fig. 4A, the level of SNARE complexes found in the starved cells was much higher than in the control cells, indicating that disassembly of SNARE complexes involved in exocytosis is inhibited under amino acid starvation conditions. To further examine the effect of amino acid deprivation on the oligomeric state of SNARE molecules, we isolated membrane extracts from control and starved CHO cells and analyzed the oligomeric states of GOS-28, a v-SNARE participating in intra-Golgi transport, and its cognate t-SNARE, syntaxin-5. Consistent with the results obtained with the PC12 cells (Fig. 4A), we observed significantly more GOS-28/syntaxin-5 SDS-resistant complexes in the starved cells (Fig. 4B), indicating that the disassembly of SNARE complexes containing GOS-28 and syntaxin-5 was impaired under these conditions. The effect of starvation on the interaction between GOS-28 and syntaxin-5 was further analyzed by co-immunoprecipitation experiments. For this purpose, agarose-protein A beads coupled to anti-syntaxin-5 or anti-GOS-28 antibodies, as indicated, were mixed with membrane extracts obtained from control or starved CHO cells, and the eluted material was subjected to Western blot analysis. The results presented in Fig. 4C demonstrate that the interaction between GOS-28 and syntaxin-5 in a complex containing NSF is increased during starvation. NSF Activity Is Inhibited in Starved Cells—The accumulation of different SNARE complexes in the starved cells may best be explained by a reduction in NSF activity. To test this hypothesis, we determined the ability of cytosolic fractions obtained from control or starved cells to dissociate endogenous SNARE complexes. Rat brain membranes were first treated with NEM to abolish intrinsic NSF activity. Next, the treated membranes were incubated with cytosol obtained from starved or control CHO cells, and the level of SNARE complexes was analyzed by Western blotting. As shown in Fig. 5, incubation of NEM-treated membranes with cytosol obtained from control cells resulted in a significant dissociation of high molecular weight SNARE complexes detected in this system. In contrast, no substantial dissociation of SNARE complexes was observed upon incubation with cytosol obtained from starved cells, suggesting that NSF activity was inhibited in this fraction. Moreover, the addition of recombinant NSF to the starved cytosol recovered most of the SNARE complex disassembly activity of this fraction, indicating that the inhibition of NSF in starved cells led to the accumulation of SNARE complexes in these cells. Notably, the recovery of the disassembly activity was not complete as compared with the disassembly activity found in the control cytosol, suggesting partial inactivation of the recombinant NSF proteins by the starved cytosolic fraction (see below). NSF Undergoes a Conformational Change in Response to Starvation—NSF is a homohexamer in which each of the protomers consists of three domains: an N-terminal domain, NSF-N, which is responsible for the interaction with the α-SNAP-SNARE complex, and two homologous ATP-binding domains, NSF-D1 and NSF-D2 (31Nagiec E.E. Bernstein A. Whiteheart S.W. J. Biol. Chem. 1995; 270: 29182-29188Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 32Whiteheart S.W. Rossnagel K. Buhrow S.A. Brunner M. Jaenicke R. Rothman J.E. J. Cell Biol. 1994; 126: 945-954Crossref PubMed Scopus (343) Google Scholar). ATP binding to the D1 domain is crucial for NSF ATPas" @default.
• W2009698855 created "2016-06-24" @default.
• W2009698855 creator A5030694848 @default.
• W2009698855 creator A5046381915 @default.
• W2009698855 creator A5066148233 @default.
• W2009698855 creator A5030860797 @default.
• W2009698855 date "2005-04-01" @default.
• W2009698855 modified "2023-10-12" @default.
• W2009698855 title "Modulation of N-Ethylmaleimide-sensitive Factor Activity upon Amino Acid Deprivation" @default.
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