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• W2109586670 abstract "When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway. When faced with nutrient deprivation, Saccharomyces cerevisiae cells enter into a nondividing resting state, known as stationary phase. The Ras/PKA (cAMP-dependent protein kinase) signaling pathway plays an important role in regulating the entry into this resting state and the subsequent survival of stationary phase cells. The survival of these resting cells is also dependent upon autophagy, a membrane trafficking pathway that is induced upon nutrient deprivation. Autophagy is responsible for targeting bulk protein and other cytoplasmic constituents to the vacuolar compartment for their ultimate degradation. The data presented here demonstrate that the Ras/PKA signaling pathway inhibits an early step in autophagy because mutants with elevated levels of Ras/PKA activity fail to accumulate transport intermediates normally associated with this process. Quantitative assays indicate that these increased levels of Ras/PKA signaling activity result in an essentially complete block to autophagy. Interestingly, Ras/PKA activity also inhibited a related process, the cytoplasm to vacuole targeting (Cvt) pathway that is responsible for the delivery of a subset of vacuolar proteins in growing cells. These data therefore indicate that the Ras/PKA signaling pathway is not regulating a switch between the autophagy and Cvt modes of transport. Instead, it is more likely that this signaling pathway is controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Finally, the data suggest that at least a portion of the Ras/PKA effects on stationary phase survival are the result of the regulation of autophagy activity by this signaling pathway. Saccharomyces cerevisiae cells respond to nutrient deprivation by arresting cell division and entering into a nondividing resting state, known as the stationary phase (1Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar, 2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar). Cells in this resting state exhibit a diminished rate of metabolic activity, an increased resistance to a variety of environmental stress conditions and an altered pattern of gene expression (1Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar, 2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar, 3Werner-Washburne M. Braun E.L. Crawford M.E. Peck V.M. Mol. Microbiol. 1996; 19: 1159-1166Crossref PubMed Scopus (183) Google Scholar). Entry into this quiescent state is essential for the long term survival of these cells as mutants that do not assume stationary phase characteristics rapidly lose viability upon nutrient limitation (1Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar, 2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar). Moreover, this budding yeast, like most other eukaryotes, spends the majority of its existence in this resting state awaiting the signals needed to trigger new rounds of cell division. This latter observation has suggested that the transitions between the mitotic cycle and these periods of quiescence might be key points of proliferative control (4Pardee A.B. Science. 1989; 246: 603-608Crossref PubMed Scopus (1854) Google Scholar, 5Varmus H. Weinberg R.A. Genes and the Biology of Cancer. Scientific American Library, New York1993Google Scholar). However, despite the importance of these resting states, relatively little is known about the mechanisms controlling the entry into, and subsequent maintenance within, this stationary phase of growth. Recent studies have identified a number of genes that are important for the long term survival of S. cerevisiae stationary phase cells (2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar, 6Howard S.C. Hester A. Herman P.K. Genetics. 2003; 165: 1059-1070Crossref PubMed Google Scholar, 7Howard S.C. Chang Y.W. Budovskaya Y.V. Herman P.K. Genetics. 2001; 159: 77-89Crossref PubMed Google Scholar, 8Chang Y.W. Howard S.C. Budovskaya Y.V. Rine J. Herman P.K. Genetics. 2001; 157: 17-26PubMed Google Scholar, 9Reinders A. Burckert N. Boller T. Wiemken A. De Virgilio C. Genes Dev. 1998; 12: 2943-2955Crossref PubMed Scopus (161) Google Scholar). Interestingly, many of these genes encode proteins important for the process of macroautophagy (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1410) Google Scholar, 11Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar). Macroautophagy (to be referred to as simply autophagy for the remainder of this report) is a degradative pathway that is responsible for delivering bulk protein and other constituents of the cytoplasm to the vacuole for degradation (11Klionsky D.J. Ohsumi Y. Annu. Rev. Cell Dev. Biol. 1999; 15: 1-32Crossref PubMed Scopus (391) Google Scholar, 12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (757) Google Scholar). This pathway is induced by nutrient deprivation and mutants that are defective for autophagy rapidly lose viability during stationary phase growth (10Tsukada M. Ohsumi Y. FEBS Lett. 1993; 333: 169-174Crossref PubMed Scopus (1410) Google Scholar, 14Abeliovich H. Klionsky D.J. Microbiol. Mol. Biol. Rev. 2001; 65: 463-479Crossref PubMed Scopus (144) Google Scholar). Autophagy begins with the encapsulation of cytoplasmic material through the formation of a double membrane structure, termed the autophagosome (12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 13Mizushima N. Ohsumi Y. Yoshimori T. Cell Struct. Funct. 2002; 27: 421-429Crossref PubMed Scopus (757) Google Scholar, 15Baba M. Osumi M. Ohsumi Y. Cell Struct. Funct. 1995; 20: 465-471Crossref PubMed Scopus (128) Google Scholar, 16Baba M. Takeshige K. Baba N. Ohsumi Y. J. Cell Biol. 1994; 124: 903-913Crossref PubMed Scopus (403) Google Scholar). Recent work has indicated that this intermediate originates from a novel cytoplasmic site, termed the pre-autophagosomal structure (12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 17Kim J. Huang W.-P. Stromhaug P.E. Klionsky D.J. J. Biol. Chem. 2002; 277: 763-773Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 18Suzuki K. Kirisako T. Kamada Y. Mizushima N. Noda T. Ohsumi Y. EMBO J. 2001; 20: 5971-5981Crossref PubMed Scopus (800) Google Scholar). The outer membrane of the autophagosome subsequently fuses with the vacuole releasing the internal vesicle, the autophagic body, into the lumen of this degradative compartment (12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 19Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (959) Google Scholar). The hydrolytic enzymes within the vacuole then degrade both the delimiting membrane and the contents of the autophagic body (19Takeshige K. Baba M. Tsuboi S. Noda T. Ohsumi Y. J. Cell Biol. 1992; 119: 301-311Crossref PubMed Scopus (959) Google Scholar, 20Kim J. Scott S.V. Klionsky D.J. Int. Rev. Cytol. 2000; 198: 153-201Crossref PubMed Google Scholar, 21Klionsky D.J. J. Biol. Chem. 1998; 273: 10807-10810Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 22Scott S.V. Baba M. Ohsumi Y. Klionsky D.J. J. Cell Biol. 1997; 138: 37-44Crossref PubMed Scopus (142) Google Scholar). When this pathway is fully induced, it is generally responsible for the majority of the protein and membrane turnover occurring in those cells (21Klionsky D.J. J. Biol. Chem. 1998; 273: 10807-10810Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23Mortimore G.E. Poso A.R. Annu. Rev. Nutr. 1987; 7: 539-564Crossref PubMed Google Scholar). Previous work has implicated the Tor signal transduction pathway in the control of both autophagy and stationary phase entry in S. cerevisiae. The inactivation of this signaling pathway results in the induction of autophagy and in a growth arrest that resembles that associated with stationary phase cells (24Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (909) Google Scholar, 25Stromhaug P.E. Klionsky D.J. Traffic. 2001; 2: 524-531Crossref PubMed Scopus (138) Google Scholar). The Tor proteins are conserved serine/threonine-specific protein kinases that appear to coordinate the overall growth rate with nutrient availability in eukaryotic cells (26Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar, 27Raught B. Gingras A.C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar). Consistent with this proposition, a wide variety of Tor pathway targets important for the control of cell growth have been identified in both yeast and other eukaryotic cells (26Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar, 27Raught B. Gingras A.C. Sonenberg N. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7037-7044Crossref PubMed Scopus (507) Google Scholar, 28Lorberg A. Hall M.N. Curr. Top Microbiol. Immunol. 2004; 279: 1-18Crossref PubMed Google Scholar, 29Shamji A.F. Nghiem P. Schreiber S.L. Mol. Cell. 2003; 12: 271-280Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Stationary phase metabolism in S. cerevisiae is also controlled by a second signaling pathway that involves the Ras proteins and the cAMP-dependent protein kinase, PKA 1The abbreviations used are: PKA, cAMP-dependent protein kinase; CPY, carboxypeptidase Y; Cvt, cytoplasm to vacuole targeting; EM, electron microscopy. (1Werner-Washburne M. Braun E. Johnston G.C. Singer R.A. Microbiol. Rev. 1993; 57: 383-401Crossref PubMed Google Scholar, 2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar, 30Broach J.R. Trends Genet. 1991; 7: 28-33Abstract Full Text PDF PubMed Scopus (170) Google Scholar). The eukaryotic Ras proteins are small, GTP-binding proteins that function as molecular switches by oscillating between an active GTP-bound form and an inactive GDP-bound form (31Lowy D.R. Willumsen B.M. Annu. Rev. Biochem. 1993; 62: 851-891Crossref PubMed Scopus (1124) Google Scholar, 32Gibbs J.B. Marshall M.S. Microbiol. Rev. 1989; 53: 171-185Crossref PubMed Google Scholar). S. cerevisiae has two Ras proteins, Ras1p and Ras2p, that directly interact with the enzyme adenylyl cyclase (33Field J. Xu H.P. Michaeli T. Ballester R. Sass P. Wigler M. Colicelli J. Science. 1990; 247: 464-467Crossref PubMed Scopus (68) Google Scholar, 34Suzuki N. Choe H.R. Nishida Y. Yamawaki-Kataoka Y. Ohnishi S. Tamaoki T. Kataoka T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 8711-8715Crossref PubMed Scopus (100) Google Scholar). This interaction results in higher intracellular levels of cAMP and ultimately in increased PKA activity (35Toda T. Cameron S. Sass P. Zoller M. Wigler M. Cell. 1987; 50: 277-287Abstract Full Text PDF PubMed Scopus (507) Google Scholar, 36Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (709) Google Scholar). A role for this pathway in the regulation of stationary phase was suggested by the phenotypes of mutants with altered levels of Ras/PKA activity. For example, mutants with constitutively elevated levels of Ras/PKA activity fail to adopt stationary phase characteristics upon nutrient limitation (36Toda T. Uno I. Ishikawa T. Powers S. Kataoka T. Broek D. Cameron S. Broach J. Matsumoto K. Wigler M. Cell. 1985; 40: 27-36Abstract Full Text PDF PubMed Scopus (709) Google Scholar). Conversely, mutations that inactivate this pathway result in a permanent stationary phase-like arrest (30Broach J.R. Trends Genet. 1991; 7: 28-33Abstract Full Text PDF PubMed Scopus (170) Google Scholar, 37Iida H. Yahara I. J. Cell Biol. 1984; 98: 1185-1193Crossref PubMed Scopus (55) Google Scholar, 38Matsumoto K. Uno I. Ishikawa T. Exp. Cell Res. 1983; 146: 151-161Crossref PubMed Scopus (95) Google Scholar). Thus, two different signaling pathways involving the Ras and Tor proteins appear to play a significant role in the control of stationary phase biology in S. cerevisiae (2Herman P.K. Curr. Opin. Microbiol. 2002; 5: 602-607Crossref PubMed Scopus (182) Google Scholar, 26Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar). We are interested in characterizing the role of the Ras/PKA signaling pathway in the regulation of autophagy. Although the nature of this regulation has not been examined in detail, a recent study has found that increased levels of Ras/PKA signaling activity result in a decrease in the autophagic activity induced by the drug, rapamycin (39Schmelzle T. Beck T. Martin D.E. Hall M.N. Mol. Cell. Biol. 2004; 24: 338-351Crossref PubMed Scopus (212) Google Scholar). Rapamycin is a potent inhibitor of Tor activity in vivo and is often used to study the consequences of Tor pathway inactivation (40Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1551) Google Scholar, 41Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (728) Google Scholar). Thus, these data were consistent with the Ras/PKA pathway having some regulatory role in the autophagy process. In this study, we examined this regulation in detail and found that Ras/PKA signaling activity controls a relatively early step in the autophagy pathway. This Ras-sensitive step likely precedes the formation of the autophagosome as these transport intermediates were not formed in cells with elevated levels of Ras/PKA signaling activity. This signaling pathway also inhibited a vacuolar trafficking pathway that is related to autophagy but functions during the log phase of growth. This cytoplasm to vacuole targeting (Cvt) pathway shares many mechanistic features with autophagy and most of the proteins required for autophagy are also required for Cvt transport (42Khalfan W.A. Klionsky D.J. Curr. Opin. Cell Biol. 2002; 14: 468-475Crossref PubMed Scopus (58) Google Scholar, 43Harding T.M. Morano K.A. Scott S.V. Klionsky D.J. J. Cell Biol. 1995; 131: 591-602Crossref PubMed Scopus (399) Google Scholar, 44Scott S.V. Hefner-Gravink A. Morano K.A. Noda T. Ohsumi Y. Klionsky D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12304-12308Crossref PubMed Scopus (215) Google Scholar, 45Harding T.M. Hefner-Gravink A. Thumm M. Klionsky D.J. J. Biol. Chem. 1996; 271: 17621-17624Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). In all, these data indicate that Ras/PKA activity is not regulating the switch between autophagy and the Cvt pathway in vivo and is instead controlling an activity that is required during the early stages of both of these membrane trafficking pathways. Growth Media—Standard Escherichia coli growth conditions and media were used throughout this study (46Miller J. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972Google Scholar). The yeast rich growth medium, YPAD, consists of 1% yeast extract, 2% Bacto-peptone, 50 mg/l adenine-HCl, and 2% glucose. The yeast YM minimal growth medium consists of 0.67% yeast nitrogen base lacking amino acids, 2% glucose and all of the growth supplements required for cell proliferation (8Chang Y.W. Howard S.C. Budovskaya Y.V. Rine J. Herman P.K. Genetics. 2001; 157: 17-26PubMed Google Scholar). The nitrogen starvation medium, SD-N, consists of 0.17% yeast nitrogen base lacking amino acids and ammonium sulfate and 2% glucose. Growth media reagents were from DIFCO. Plasmid Constructions—The construction of the MET3-RAS2val19 integrating plasmid, pPHY446, was described previously (7Howard S.C. Chang Y.W. Budovskaya Y.V. Herman P.K. Genetics. 2001; 159: 77-89Crossref PubMed Google Scholar). The integration of this allele was directed to the RAS2 locus by digesting this plasmid with XmnI prior to transformation of yeast cells. For the high copy number TPK1 plasmid, pPHY2056, a 1.7-kb PCR fragment containing the TPK1 gene was cloned into the pRS426 plasmid (47Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar, 48Christianson T.W. Sikorski R.S. Dante M. Shero J.H. Hieter P. Gene (Amst.). 1992; 110: 119-122Crossref PubMed Scopus (1434) Google Scholar). A dominant negative allele of RAS2, known as RAS2ala22, was generated by a site-directed mutagenesis of a RAS2 plasmid, pJW82, that was kindly provided by Dr. Jasper Rine. The pJW82 plasmid has RAS2 placed under the control of the inducible promoter from the GAL1 gene. The site-directed mutagenesis was performed with the Transformer mutagenesis kit (Clontech) and resulted in the substitution of an alanine residue for the glycine normally found at position 22 of Ras2p. The resulting plasmid was called pPHY2128. The effects of this dominant negative allele have been described previously (49Powers S. O'Neill K. Wigler M. Mol. Cell. Biol. 1989; 9: 390-395Crossref PubMed Scopus (131) Google Scholar, 50Haney S.A. Broach J.R. J. Biol. Chem. 1994; 269: 16541-16548Abstract Full Text PDF PubMed Google Scholar). For the Ape1p overexpression experiments, the APE1 coding sequences were placed under the control of the inducible promoter from the yeast CUP1 gene; CUP1 encodes a copper-binding metallothionein (51Thiele D.J. Hamer D.H. Mol. Cell. Biol. 1986; 6: 1158-1163Crossref PubMed Scopus (73) Google Scholar, 52Winge D.R. Nielson K.B. Gray W.R. Hamer D.H. J. Biol. Chem. 1985; 260: 14464-14470Abstract Full Text PDF PubMed Google Scholar). Expression from the CUP1 promoter was induced by the addition of 100 μm CuSO4 to the growth medium. Yeast Strain Construction and Growth Conditions—The strains used in this study are listed in Table I. Unless otherwise noted, strains were from our laboratory collections or were derived during the course of this work. Standard yeast genetic methods were used for the construction of all strains (53Kaiser C. Michaelis S. Mitchell A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar).Table IYeast strains used in this studyStrainGenotypeAliasRef.PHY3513TN125, RAS2::HIS3-RAS2val19PHY3992TVY1, RAS2::HIS3-RAS2val19PHY3993TDY2, RAS2::HIS3-RAS2val19TDY2MATα his3-Δ200 leu2-3,112 lys2-80 trp1-Δ901 ura3-52 suc2-Δ9 vam3Δ::LEU2 p(vam3ts)PHY367174Darsow T. Rieder S.E. Emr S.D. J. Cell Biol. 1997; 138: 517-529Crossref PubMed Scopus (297) Google ScholarTN125MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho8Δ60PHY280124Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (909) Google ScholarTVY1MATα his3-Δ200 leu2-3,112 lys2-80 trp1-Δ901 ura3-52 suc2-Δ9 pep4Δ::LEU2PHY367086Gerhardt B. Kordas T.J. Thompson C.M. Patel P. Vida T. J. Biol. Chem. 1998; 273: 15818-15829Abstract Full Text Full Text PDF PubMed Scopus (50) Google ScholarYYK126MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho8Δ60 atg1Δ::LEU2PHY280224Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (909) Google ScholarYYK130MATa ade2 his3 leu2 lys2 trp1 ura3 pho8::pho8Δ60 atg13Δ::LEU2PHY280324Kamada Y. Funakoshi T. Shintani T. Nagano K. Ohsumi M. Ohsumi Y. J. Cell Biol. 2000; 150: 1507-1513Crossref PubMed Scopus (909) Google Scholar Open table in a new tab Strains carrying the MET3-RAS2val19 allele were grown in medium containing 500 μm methionine to keep the MET3 promoter in its repressed state. RAS2val19 expression was induced by transferring these cells to medium that lacks methionine (7Howard S.C. Chang Y.W. Budovskaya Y.V. Herman P.K. Genetics. 2001; 159: 77-89Crossref PubMed Google Scholar, 54Howard S.C. Budovskaya Y.V. Chang Y.W. Herman P.K. J. Biol. Chem. 2002; 277: 19488-19497Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). For the experiments with the dominant negative allele, GAL1-RAS2ala22, a wild-type strain, TN125, was transformed with either pPHY2128 or a control vector, pRS416. These strains were grown to mid-log phase in YM-glucose minimal medium and were transferred to YM medium containing 5% galactose and 2% raffinose. Culture samples were collected at the indicated time intervals and alkaline phosphatase assays were performed to assess autophagy activity, as described below. Alkaline Phosphatase-based Autophagy Assays—Autophagy levels were measured with an alkaline phosphatase-based assay that has been described previously (55Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). In general, autophagy was induced by transferring cells to a medium that lacks a nitrogen source, SD-N. Alkaline phosphatase levels were typically determined for cells that had been incubated in SD-N medium for 0 and 15 h at 30 °C. The increase in alkaline phosphatase activity that was observed following the period of starvation was a direct measure of the autophagy activity present in those cells. For the assays, the cells were resuspended in 200 μl of the assay buffer (250 mm Tris-SO4, pH 9.4, 10 mm MgSO4, 10 μm ZnSO4) and disrupted by vortexing with glass beads. The cell lysates were clarified by centrifugation at 10,000 × g for 5 min, and 50 μlofthe resulting protein extract was added to a tube containing 500 μl of assay buffer and 50 μl of a 1 mg/ml solution of p-nitrophenylphosphate. This reaction mix was incubated for 30 min at 35 °C, and the reaction was then stopped by the addition of 500 μlof2 m glycine-NaOH, pH 11. The absorbance at 405 nm of the resulting solution was then measured. One unit of activity is defined as the release of 1 μmol of p-nitrophenyl/min/mg of protein. The protein concentrations in the cell extracts were determined with a bicinchoninic acid protein assay kit (Sigma). For the rapamycin experiments, autophagy assays were performed on cells that had been treated with 0.2 μg/ml rapamycin for 0, 1, or 3 h at 30 °C. Western Immunoblotting and Immunoprecipitations—For the Western immunoblots, protein extracts were prepared by a glass-beading method described previously (56Klekamp M.S. Weil P.A. J. Biol. Chem. 1982; 257: 8432-8441Abstract Full Text PDF PubMed Google Scholar, 57Budovskaya Y.V. Hama H. DeWald D.B. Herman P.K. J. Biol. Chem. 2002; 277: 287-294Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The resulting protein extracts were separated on SDS-polyacrylamide gels and transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences) at 4 °C. The membranes were then probed with either anti-Ape1p (58Klionsky D.J. Cueva R. Yaver D.S. J. Cell Biol. 1992; 119: 287-299Crossref PubMed Scopus (306) Google Scholar), anti-Pgk1p, or anti-Atg13p antisera, and the immunoreactive proteins were detected with anti-rabbit IgG (Amersham Biosciences) used at a dilution of 1:3000. The Supersignal chemiluminescent substrate (Pierce) was subsequently used to illuminate the reactive bands. The carboxypeptidase Y (CPY) immunoprecipitation experiments were performed as described (57Budovskaya Y.V. Hama H. DeWald D.B. Herman P.K. J. Biol. Chem. 2002; 277: 287-294Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 59Herman P.K. Stack J.H. DeModena J.A. Emr S.D. Cell. 1991; 64: 425-437Abstract Full Text PDF PubMed Scopus (157) Google Scholar). Electron Microscopy—The pep4Δ (TVY1) and vam3ts (TDY2) strains carrying either a control vector or an integrated version of the MET3-RAS2val19 construct, were grown overnight in YM-glucose medium containing 500 μm methionine. The cells were washed twice with water and transferred to a YM-glucose medium lacking methionine for 4 h to induce RAS2val19 expression. The cells were then washed twice with water and transferred to SD-N for 3 h. All incubations for the pep4Δ strains were performed at 30 °C. For the vam3ts strains, the incubations in YM-glucose media were performed at 26 °C, whereas the incubations in the SD-N medium were done at 37 °C to inactivate the temperature-sensitive Vam3p present. Cells were fixed with permanganate, dehydrated with acetone, and embedded with Spurr's resin as described (60Kaiser C.A. Schekman R. Cell. 1990; 61: 723-733Abstract Full Text PDF PubMed Scopus (545) Google Scholar). 70-nm sections were mounted on nickel grids, and stained with uranyl acetate and lead citrate to be finally imaged with a Philips CM-100 transmission electron microscope. Elevated Levels of Ras/PKA Signaling Activity Inhibited Autophagy—We were interested in examining the role of the Ras/PKA pathway in the regulation of autophagy. For our initial experiments, autophagy activity was measured with an assay that analyzes the vacuolar delivery of a cytoplasmic variant of the Pho8p alkaline phosphatase following a shift to conditions of nitrogen starvation (55Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). The wild-type Pho8p is synthesized as an inactive zymogen that is delivered to the vacuolar compartment via the traditional secretory pathway (61Klionsky D.J. Emr S.D. EMBO J. 1989; 8: 2241-2250Crossref PubMed Scopus (235) Google Scholar, 62Klionsky D.J. Emr S.D. J. Biol. Chem. 1990; 265: 5349-5352Abstract Full Text PDF PubMed Google Scholar). Upon arrival in the vacuole lumen, this zymogen is activated by a proteolytic cleavage that removes a C-terminal propeptide (61Klionsky D.J. Emr S.D. EMBO J. 1989; 8: 2241-2250Crossref PubMed Scopus (235) Google Scholar). The cells used for the autophagy assay express a Pho8p variant, Pho8pΔ60, that lacks a transmembrane domain that functions as an internal signal sequence and normally directs this protein into the secretory pathway (55Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar, 61Klionsky D.J. Emr S.D. EMBO J. 1989; 8: 2241-2250Crossref PubMed Scopus (235) Google Scholar). As a result, Pho8pΔ60 is found in the cytosol in its inactive, precursor form. Following the induction of autophagy, this protein is packaged into autophagosomes and delivered to the vacuole where it can be activated (12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 55Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). Previous work has indicated that the levels of alkaline phosphatase activity present are proportional to the amount of autophagy occurring in these cells (12Noda T. Suzuki K. Ohsumi Y. Trends Cell Biol. 2002; 12: 231-235Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 55Noda T. Matsuura A. Wada Y. Ohsumi Y. Biochem. Biophys. Res. Commun. 1995; 210: 126-132Crossref PubMed Scopus (295) Google Scholar). The Pho8pΔ60 assay was used to examine the effect that elevated levels of Ras/PKA signaling activity would have on the autophagy process. To increase Ras/PKA activity, a dominant hyperactive allele of RAS2, known as RAS2val19, was introduced into the assay strain. The presence of this allele results in constitutively elevated levels of Ras/PKA signaling activity (30Broach J.R. Trends Genet. 1991; 7: 28-33Abstract Full Text PDF PubMed Scopus (170) Google Scholar," @default.
• W2109586670 created "2016-06-24" @default.
• W2109586670 creator A5004778008 @default.
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• W2109586670 date "2004-05-01" @default.
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• W2109586670 title "The Ras/cAMP-dependent Protein Kinase Signaling Pathway Regulates an Early Step of the Autophagy Process in Saccharomyces cerevisiae" @default.
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• W2109586670 doi "https://doi.org/10.1074/jbc.m400272200" @default.
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