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• W2015468911 abstract "The export of bulk poly(A)+ mRNA is blocked under heat-shocked (42 °C) conditions in Saccharomyces cerevisiae. We found that an mRNA export factor Gle2p rapidly dissociated from the nuclear envelope and diffused into the cytoplasm at 42 °C. However, in exponential phase cells pretreated with mild heat stress (37 °C for 1 h), Gle2p did not dissociate at 42 °C, and the export of bulk poly(A)+ mRNA continued. Cells in stationary phase also continued with the export of bulk poly(A)+ mRNA at 42 °C without the dissociation of Gle2p from the nuclear envelope. The dissociation of Gle2p was caused by increased membrane fluidity and correlated closely with blocking of the export of bulk poly(A)+ mRNA. Furthermore, the mutants gle2Δ and rip1Δ could not induce such an adaptation of the export of bulk poly(A)+ mRNA to heat shock. Our findings indicate that Gle2p plays a crucial role in mRNA export especially under heat-shocked conditions. Our findings also indicate that the nuclear pore complexes that Gle2p constitutes need to be stabilized for the adaptation and that the increased membrane integrity caused by treatment with mild heat stress or by survival in stationary phase is likely to contribute to the stabilization of the association between Gle2p and the nuclear pore complexes. The export of bulk poly(A)+ mRNA is blocked under heat-shocked (42 °C) conditions in Saccharomyces cerevisiae. We found that an mRNA export factor Gle2p rapidly dissociated from the nuclear envelope and diffused into the cytoplasm at 42 °C. However, in exponential phase cells pretreated with mild heat stress (37 °C for 1 h), Gle2p did not dissociate at 42 °C, and the export of bulk poly(A)+ mRNA continued. Cells in stationary phase also continued with the export of bulk poly(A)+ mRNA at 42 °C without the dissociation of Gle2p from the nuclear envelope. The dissociation of Gle2p was caused by increased membrane fluidity and correlated closely with blocking of the export of bulk poly(A)+ mRNA. Furthermore, the mutants gle2Δ and rip1Δ could not induce such an adaptation of the export of bulk poly(A)+ mRNA to heat shock. Our findings indicate that Gle2p plays a crucial role in mRNA export especially under heat-shocked conditions. Our findings also indicate that the nuclear pore complexes that Gle2p constitutes need to be stabilized for the adaptation and that the increased membrane integrity caused by treatment with mild heat stress or by survival in stationary phase is likely to contribute to the stabilization of the association between Gle2p and the nuclear pore complexes. When exposed to various forms of stress, cells show adaptive responses such as changes in patterns of gene expression, in metabolism, and in other cellular processes. Adaptive response mechanisms aim to repair molecular damage and to protect cells against potentially adverse effects of stress, resulting in an increase in stress tolerance. Therefore, yeast cells pretreated with a comparatively mild and sublethal stress show increased resistance to subsequent lethal stress, since diverse adaptive responses are induced during the pretreatment (1Jamieson D.J. J. Bacteriol. 1992; 174: 6678-6681Crossref PubMed Google Scholar, 2Izawa S. Inoue Y. Kimura A. Biochem. J. 1996; 320: 61-67Crossref PubMed Scopus (203) Google Scholar, 3Piper P. Hohmann S. Mager W.H. Yeast Stress Response. Springer-Verlag, Heidelberg, Germany1997: 75-99Google Scholar, 4Cotto J.J. Morimoto R.I. Biochem. Soc. Symp. 1999; 64: 105-118PubMed Google Scholar). This phenomenon is termed adaptation. It is well known that the expression of heat shock proteins is induced during pretreatment with mild heat shock and contributes to an increased resistance to severe heat shock (3Piper P. Hohmann S. Mager W.H. Yeast Stress Response. Springer-Verlag, Heidelberg, Germany1997: 75-99Google Scholar, 4Cotto J.J. Morimoto R.I. Biochem. Soc. Symp. 1999; 64: 105-118PubMed Google Scholar, 5Miller M.J. Xuong N.H. Geiduschek E.P. J. Bacteriol. 1982; 151: 311-327Crossref PubMed Google Scholar, 6Pirkkala L. Nykanen P. Sistonen L. FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (825) Google Scholar). Heat shock proteins play important roles in protecting other proteins against denaturation and in restoring their biological activities disrupted by stress (3Piper P. Hohmann S. Mager W.H. Yeast Stress Response. Springer-Verlag, Heidelberg, Germany1997: 75-99Google Scholar, 7Craig E.A. Gambill B.D. Nelson R.J. Microbiol. Rev. 1993; 57: 402-414Crossref PubMed Google Scholar). Under stressed conditions, yeast cells undergo changes not only in transcriptional patterns but also in the types of mRNA that are nuclear exported in order to adapt rapidly to the stress. It is known that both heat shock and ethanol affect the export of mRNA. Under heat-shocked (42 °C) conditions, yeast cells shut down the synthesis of most proteins with the exception of stress-responsive proteins such as heat shock proteins. This effect is caused in part by selective mRNA export. Stress-induced transcripts such as SSA4 encoding one of the Hsp70 proteins are efficiently exported through nuclear pore complexes (NPCs), 1The abbreviations used are: NPC, nuclear pore complex; hs mRNA, heat shock mRNA; GFP, green fluorescent protein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; BA, benzyl alcohol; DAPI, 4′,6-diaminidino-2-phenylindole. whereas bulk poly(A)+ mRNA accumulates in the nucleus under heat-shocked conditions (8Saaverda C. Tung K.S. Amberg D.C. Hopper A.K. Cole C.N. Genes Dev. 1996; 10: 1608-1620Crossref PubMed Scopus (138) Google Scholar, 9Krebber H. Taura T. Lee M.S. Silver P.A. Genes Dev. 1999; 13: 1994-2004Crossref PubMed Scopus (67) Google Scholar). However, pretreatment of cells with a mild heat shock before a severe heat shock protects the mRNA export machinery and allows mRNA export to proceed unimpeded under subsequent severe heat-shocked conditions (10Tani T. Derby R.J. Hiraoka Y. Spector D.L. Mol. Biol. Cell. 1995; 6: 1515-1534Crossref PubMed Scopus (64) Google Scholar). In Saccharomyces cerevisiae, the shut-off of bulk poly (A)+ mRNA export under stressed conditions involves the dissociation of Npl3p, a heterogeneous nuclear ribonucleoprotein, from mRNA (9Krebber H. Taura T. Lee M.S. Silver P.A. Genes Dev. 1999; 13: 1994-2004Crossref PubMed Scopus (67) Google Scholar). Additionally, the nucleoporin Rip1p/Nup42p was proposed to play an important role in the export of heat shock mRNA (hs mRNA) under heat-shocked conditions (11Saavedra C.A. Hammell C.M. Heath C.V. Cole C.N. Genes Dev. 1997; 11: 2845-2856Crossref PubMed Scopus (113) Google Scholar, 12Stutz F. Kantor J. Zhang D. McCarthy T. Neville M. Rosbash M. Genes Dev. 1997; 11: 2857-2868Crossref PubMed Scopus (83) Google Scholar). Initially, it was proposed that hs mRNA is exported through a specific pathway defined by the nucleoporin Rip1p/Nup42p (11Saavedra C.A. Hammell C.M. Heath C.V. Cole C.N. Genes Dev. 1997; 11: 2845-2856Crossref PubMed Scopus (113) Google Scholar, 12Stutz F. Kantor J. Zhang D. McCarthy T. Neville M. Rosbash M. Genes Dev. 1997; 11: 2857-2868Crossref PubMed Scopus (83) Google Scholar). However, it has been recently reported that Rip1p also participates in the export of non-hs mRNA at 42 °C (13Vainberg I.E. Dower K. Rosbash M. Mol. Cell. Biol. 2000; 20: 3996-4005Crossref PubMed Scopus (42) Google Scholar). There is also a report that other general mRNA export factors are involved in the export of hs mRNA at elevated temperatures, suggesting that hs mRNA and non-hs mRNA are exported through similar pathways (13Vainberg I.E. Dower K. Rosbash M. Mol. Cell. Biol. 2000; 20: 3996-4005Crossref PubMed Scopus (42) Google Scholar, 14Hurt E. Strässer K. Segref A. Bailer S. Schlaich N. Presutti C. Tollervey D. Jensen R. J. Biol. Chem. 2000; 275: 8361-8368Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The pathways of mRNA export under stressed conditions seem to be quite complex and still controversial. It is reasonable to imagine that changes in the processing of pre-mRNA would occur under stressed conditions, since the quality of mRNA affects competency for export (15Proudfoot N. Trends Biochem. Sci. 2000; 25: 290-293Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 16Lei E.P. Silver P.A. Genes Dev. 2002; 16: 2761-2766Crossref PubMed Scopus (94) Google Scholar, 17Strässer K. Masuda S. Mason P. Pfannstiel J. Oppizzi M. Rodriguez-Navarro S. Rondón A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (640) Google Scholar, 18Reed R. Curr. Opin. Cell Biol. 2003; 15: 326-331Crossref PubMed Scopus (205) Google Scholar). It has been reported that splicing is blocked following heat shock (19Vogel J.L. Parsell D.A. Lindquist S. Curr. Biol. 1995; 5: 306-317Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Appropriate processing steps such as splicing, 5′ capping, 3′ cleavage, and polyadenylation are necessary for the efficient nuclear export of mRNA (7Craig E.A. Gambill B.D. Nelson R.J. Microbiol. Rev. 1993; 57: 402-414Crossref PubMed Google Scholar, 16Lei E.P. Silver P.A. Genes Dev. 2002; 16: 2761-2766Crossref PubMed Scopus (94) Google Scholar, 20Bentley D. Curr. Opin. Cell Biol. 1999; 11: 347-351Crossref PubMed Scopus (196) Google Scholar, 21Brodsky A.S. Silver P.A. RNA. 2000; 6: 1737-1749Crossref PubMed Scopus (155) Google Scholar, 22Hilleren P. McCarthy T. Rosbash M. Parker R. Jensen T.H. Nature. 2001; 413: 538-542Crossref PubMed Scopus (294) Google Scholar, 23Hammell C.M. Gross S. Zenklusen D. Heath C.V. Stutz F. Moore C. Cole C.N. Mol. Cell. Biol. 2002; 22: 6441-6457Crossref PubMed Scopus (112) Google Scholar). Both hyperadenylation and defects in polyadenylation of the 3′-end of mRNA cause the blocking of mRNA export and the apparent accumulation of pre-mRNA at the site of transcription (21Brodsky A.S. Silver P.A. RNA. 2000; 6: 1737-1749Crossref PubMed Scopus (155) Google Scholar, 24Jensen T.H. Patricio K. McCarthy T. Rosbash M. Mol. Cell. 2001; 7: 887-898Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Vice versa, several mutant strains defective in mRNA export also show hyperadenylation of the 3′-end of mRNA (25Hilleren P. Parker R. RNA. 2001; 7: 753-764Crossref PubMed Scopus (76) Google Scholar). Changes in pre-mRNA quality caused by processing factors may regulate the export competency of mRNA under conditions of stress. Another possibility is that mRNA export factors have an effect on selective mRNA export under stressed conditions. In S. cerevisiae, matured mRNA is exported as messenger ribonucleoprotein complexes by mRNA export factors including RNA-binding proteins (Mex67p, Sub2p, Yra1p, Yra2p, and Npl3p), nucleoporins and NPC-associated proteins (Gle1p, Gle2p, Mtr2p, Rip1p/Nup42p, and Rat7p/Nup159p), and DEAD box RNA helicase Rat8p/Dbp5p. Several reviews describe well the functions and interactions of yeast mRNA export factors (26Cole C.N. Fantes P. Begges J. The Yeast Nucleus. Oxford University Press, New York2000: 276-311Google Scholar, 27Zenklusen D. Stutz F. FEBS Lett. 2001; 498: 150-156Crossref PubMed Scopus (60) Google Scholar, 28Lei E.P. Silver P.A. Dev. Cell. 2002; 2: 261-272Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). It is still not clear how NPCs and mRNA export factors alter their functions in response to stress, except for the response of Npl3p and Hrp1p (9Krebber H. Taura T. Lee M.S. Silver P.A. Genes Dev. 1999; 13: 1994-2004Crossref PubMed Scopus (67) Google Scholar, 29Henry M.F. Mandel D. Routson V. Henry P.A. Mol. Biol. Cell. 2003; 14: 3929-3941Crossref PubMed Google Scholar). Therefore, it is quite difficult at present to estimate the contribution of mRNA export factors to selective mRNA export. The stress responses of mRNA processing factors and export factors still remain to be clarified. In this study, we investigated the adaptive responses of mRNA export factors to heat shock. We found that most of the Gle2p dissociated from the NPCs and diffused into the cytoplasm under heat-shocked conditions, correlating well with the blocking of bulk poly(A)+ mRNA export. Gle2p associates with the nuclear pores through interaction with Rip1p (30Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar) and Nup116p (31Bailer S.M. Siniossoglou S. Podtelejnikov A. Helwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar). It has been reported that Gle2p plays a role in mRNA export in the NPCs (30Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar). Intriguingly, pretreatment with mild heat stress precluded the dissociation of Gle2p and the accumulation of mRNA in the nucleus under subsequent severe heat-shocked conditions. Stationary phase cells also exported bulk poly(A)+ mRNA without the dissociation of Gle2p under stressed conditions. Changes in the intracellular localization of Gle2p correlated closely with this adaptation of the export of bulk poly(A)+ mRNA. Here we suggest the possibility that Gle2p regulates the export of bulk poly(A)+ mRNA under conditions of stress through changes in its localization. The S. cerevisiae strains used in this study were W303–1A (MATahis3–11, 15 leu2–3, 112 trp1–1 ade2–1 ura3–1 can1–100), SWY1920 (MATaGLE2-GFP:HIS3 ade2–1::ADE2 his3–11, 15 leu2–3, 112 trp1–1 ura3–1 can1–100) (32Strawn L.A. Shen T. Wente S.R. J. Biol. Chem. 2001; 276: 6445-6452Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), SWY1226 (MATα his3–11, 15 leu2–3, 112 trp1–1 ade2–1 ura3–1 can1–100 gle2::HIS3) (33Bucci M. Wente S.R. Mol. Biol. Cell. 1998; 9: 2439-2461Crossref PubMed Scopus (42) Google Scholar), and FSY17 (MATahis3–11, 15 leu2–3, 112 trp1–1 ade2–1 ura3–1 rip1::KanR) (12Stutz F. Kantor J. Zhang D. McCarthy T. Neville M. Rosbash M. Genes Dev. 1997; 11: 2857-2868Crossref PubMed Scopus (83) Google Scholar). The SWY strains and FSY17 were donated by Dr. S. R. Wente and Dr. F. Stutz, respectively. Cells were cultured in 50 ml of SD minimal medium (2% glucose and 0.67% yeast nitrogen base without amino acids, pH 5.5) with appropriate amino acids and bases at 28 °C with reciprocal shaking in 300-ml Erlenmeyer flasks. Exponential phase cells were prepared by culturing until an A610 of 0.5–0.7, and stationary phase cells were prepared by culturing for over 48 h. A medium supplemented with palmitic acid was prepared with 1% Niaproof 4 (tergitol) to solubilize the fatty acid (34Stukey J.E. McDonough V.M. Martin C.E. J. Biol. Chem. 1989; 264: 16537-16544Abstract Full Text PDF PubMed Google Scholar). Palmitic acid-enriched cells were prepared as described by Mizoguchi and Hara (35Mizoguchi H. Hara S. J. Ferment. Bioeng. 1996; 81: 406-411Crossref Scopus (33) Google Scholar, 36Mizoguchi H. Hara S. J. Ferment. Bioeng. 1997; 83: 12-16Crossref Scopus (24) Google Scholar). To induce adaptation to heat shock stress at 42 °C, cells in exponential phase at 28 °C were immediately transferred into a water bath (37 °C) and then incubated with shaking at 37 °C for 1 h. YIp-GLE1-GFP—A 1698-bp fragment encoding the open reading frame of GLE1 was amplified using the primers 5′-CTAGATAATGCTAAGAGCTCAAATGCGACA-3′ and 5′-GTTCAGAATTTTCTCTAGAGACATTTCCGG-3′. The amplicon was digested with SacI/XbaI and cloned into the SacI/NheI sites of pPS1630 (37Kahana J.A. Schlenstedt G. Evanchuk D.M. Geiser J.R. Hoyt M.A. Silver P.A. Mol. Biol. Cell. 1998; 9: 1741-1756Crossref PubMed Scopus (103) Google Scholar) to construct YIp-GLE1-GFP. To integrate the GLE1-GFP gene at the chromosomal GLE1 locus, YIp-GLE1-GFP was linearized by NheI and introduced into yeast cells. YIp-GLE2-GFP—A 999-bp fragment encoding the GLE2 was amplified using the primers 5′-CGGATATTGCGTTTTCACCGCATCTAGATT-3′ and 5′-AGCTATCCGAAGAACGAATCTCGAGTTTTC-3. The amplicon was digested with XhoI/XbaI and cloned into the XhoI/XbaI sites of pPS1630 to construct YIp-GLE2-GFP. To integrate the GLE2-GFP gene at the chromosomal GLE2 locus, YIp-GLE2-GFP was linearized by ClaI and introduced into yeast cells. YIp-NUP116-GFP—A 1215-bp fragment encoding the open reading frame of NUP116 was amplified using 5′-TTCCAACGGTTCAACGGGCCTGTTTGGTAG-3′ and 5′-AACTCAGGTCTGCTCTCGAGCGTGGTTTAC-3′. The amplicon was digested with SpeI/XhoI and cloned into the SpeI/XhoI sites of pPS1630 to construct YIp-NUP116-GFP. To integrate the NUP116-GFP gene at the chromosomal NUP116 locus, YIp-NUP116-GFP was linearized by EcoRI and introduced into yeast cells. pKW-GLE2-GFP—A 1852-bp fragment encoding the promoter region and open reading frame of GLE2 was amplified using 5′-GACCGGATTACGAGCTCAATTTACTGGATA-3′ and 5′-CCGAAGAACGAATTTCACCAGATCTTTTTC-3′. The amplicon was digested with SacI/BglII and cloned into the SacI/BamHI sites of pKW430 (38Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar) to construct pKW-GLE2-GFP. pCS835 (GFP-RAT8) was donated by Dr. C. N. Cole (39Snay-Hodge C.A. Colot H.V. Goldstein A.L. Cole C.N. EMBO J. 1998; 17: 2663-2676Crossref PubMed Scopus (229) Google Scholar). pRS315-GFP-MTR2 was provided by Dr. E. Hurt (40Santos-Rosa H. Moreno H. Simos G. Segref A. Fahrenkrog B.F.A. Pante N. Hurt E. Mol. Cell. Biol. 1998; 18: 6826-6838Crossref PubMed Scopus (222) Google Scholar). pTS-RIP1-GFP was donated by Dr Y. Kikuchi (41Takahashi Y. Mizoi J. Toh-e A. Kikuchi Y. J. Biochem. (Tokyo). 2000; 128: 723-725Crossref PubMed Scopus (70) Google Scholar). pFS2146 (pHA-YRA1) and pFS2262 (pMyc-YRA2) were provided by Dr. F. Stutz (42Zenklusen D. Vinciguerra P. Strahm Y. Stutz F. Mol. Cell. Biol. 2001; 21: 4219-4232Crossref PubMed Scopus (116) Google Scholar). The oligo(dT)50 probe was labeled at its 3′-end with digoxigenin using a DIG Oligonucleotide Tailing Kit (Roche Applied Science). In situ hybridization assays to detect poly(A)+ mRNA were performed as described previously (43Amberg D.C. Goldstein A.L. Cole C. Genes Dev. 1992; 6: 1173-1189Crossref PubMed Scopus (302) Google Scholar, 44Long R.M. Elliott D.J. Stutz F. Rosbash M. Singer R.H. RNA. 1995; 1: 1071-1078PubMed Google Scholar). Cells pretreated at 37 °C and cells without pretreatment were collected and washed and then transferred to prewarmed fresh SD medium (42 °C). After incubation for 10 min at 42 °C, 10 μl of [35S]methionine/cysteine solution (Pro-mix [35S] in vitro cell labeling mix; Amersham Biosciences) was added per 10 ml of medium, and cells were labeled for 20 min at 42 °C. Subsequently, protein extract was prepared immediately by the method of Blomberg (45Blomberg A. Methods Enzymol. 2002; 350: 559-584Crossref PubMed Scopus (20) Google Scholar). Immobilized polyacrylamide gel dry strips (11 cm) with pH 4.0–7.0 gradients (Bio-Rad) were used for the first dimension separation. Soluble protein from whole-cell lysates was mixed with rehydration buffer (8 m urea, 2% CHAPS, 115 mm dithiothreitol, 0.1% Bio-Lytes, and 0.001% bromphenol blue), and 250 μl of mixed solution (80 μg of protein/ml) was added to individual lanes of a rehydration tray (BioRad). The strips were allowed to rehydrate at 20 °C for 12 h. The samples were run at 250 V for 15 min, then the voltage was raised to 8000 V over a period of 3 h and kept at 8000 V for 4.5 h in a PROTEAN IEF Cell (Bio-Rad). Subsequently, the immobilized polyacrylamide gel strips were reequilibrated in equilibration buffer I (6 m urea, 2% SDS, 0.375 m Tris-HCl, pH 8.8, 20% glycerol, and 130 mm dithiothreitol) for 15 min and in equilibration buffer II (6 m urea, 2% SDS, 0.375 m Tris-HCl, pH 8.8, 20% glycerol, and 135 mm iodoacetamide) for 15 min at 20 °C. Following reequilibration, the strips were subjected to SDS-PAGE using 12.5% polyacrylamide gels. Yeast cells were converted to spheroplasts by incubation with 10 mg/ml Zymolyase 20T (Seikagaku Co., Tokyo, Japan) in 100 mm potassium phosphate buffer with 1.2 m sorbitol (pH 7.5). Cytoplasmic fraction and nuclei fraction were prepared from the spheroplasts by the method of Bécam et al. (46Bécam A.-M. Nasr F. Racki W.J. Zagulski M. Herbert C.J. Mol. Genet. Genomics. 2001; 266: 454-462Crossref PubMed Scopus (48) Google Scholar). Each fraction (20 μg of protein) was subjected to SDS-PAGE, and proteins were electrically transferred onto polyvinylidene fluoride membrane (Immobilon-P; Millipore Corp., Bedford, MA). Nop1p and Ena1p were used as a nuclear protein marker and a cytoplasmic protein marker, respectively. Anti-Nop1p antibody and anti-Ena1p antibody were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). The secondary antibodies and anti-GFP antibody were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Change in the Localization of Gle2p under Stressed Conditions—We first investigated the localization of mRNA export factors under heat-shocked conditions (42 °C) in exponential phase cells, using various GFP fusion proteins and immunofluorescence techniques. The intracellular localization of most of the export factors we investigated did not change. Rip1p and Nup116p remained at the nuclear envelope following heat shock (Fig. 1A). The distribution of other factors such as Rat7p, Rat8p, Yra1p, Yra2p, Gle1p, and Mtr2p did not change either (data not shown). However, the localization of Gle2p clearly changed under heat-shocked conditions. Gle2p is one of the constituents of NPCs, and Gle2p-GFP usually shows a fluorescent signal around the nuclear rim with a punctate pattern of expression (30Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar), a finding we reconfirmed here (Fig. 1A). Although a certain amount of Gle2p-GFP still remained around the nuclear rim, most was dissociated from the nuclear envelope and moved into the cytoplasm under heat-shocked conditions (Fig. 1A). The dissociation of Gle2p from the nucleus was verified by cell fractionation experiments (Fig. 1B). Gle2p-GFP was detected by immunoblotting in the nuclei fraction but not in the cytoplasmic fraction of cells without stress treatment (Fig. 1B, lanes 1 and 2). However, in cells treated with heat shock at 42 °C, Gle2p-GFP was detected in both fractions (lanes 3 and 4). Compared with nonstressed cells, the level of Gle2p-GFP in the nuclei fraction was decreased in these cells (lanes 2 and 4). The localization of Gle2p was changed quickly (within 5 min) by the heat shock. We examined the levels of total Gle2p-GFP by Western blotting analysis using anti-GFP-antibody, but found no significant change under the conditions (data not shown). In the analysis in Fig. 1, the GLE2-GFP gene was integrated into the genomic DNA (33Bucci M. Wente S.R. Mol. Biol. Cell. 1998; 9: 2439-2461Crossref PubMed Scopus (42) Google Scholar). This strain (SWY1920) did not show the phenotypes of the gle2Δ mutant such as slow growth, indicating that this Gle2p-GFP was functional. We confirmed that Gle2p-GFP is functional using pKW-GLE2-GFP. A gle2Δ mutant (SWY1226) carrying pKW-GLE2-GFP recovered the growth rate, and Gle2p-GFP dissociated from the nuclear envelope in this mutant at 42 °C (data not shown). We next investigated the effects of other forms of stress. Ethanol stress (10%, v/v) as well as heat shock caused changes in the localization of Gle2p (Fig. 1A), although osmotic stress (Fig. 1, A and B, lanes 7 and 8) and treatment with various drugs such as tunicamycin and diamide (data not shown) did not. The distribution of Rip1p-GFP and Nup116p-GFP did not change under conditions of ethanol stress (Fig. 1A). Ethanol stress also induced a rapid change in the localization of Gle2p-GFP (within 5 min). It is known that heat shock and 10% ethanol cause a blocking of the export of bulk poly(A)+ mRNA (8Saaverda C. Tung K.S. Amberg D.C. Hopper A.K. Cole C.N. Genes Dev. 1996; 10: 1608-1620Crossref PubMed Scopus (138) Google Scholar, 9Krebber H. Taura T. Lee M.S. Silver P.A. Genes Dev. 1999; 13: 1994-2004Crossref PubMed Scopus (67) Google Scholar). Therefore, we further investigated the concentration-dependent effects of ethanol on the changes in the localization of Gle2p and the export of bulk poly(A)+ mRNA. As shown in Fig. 2, 6% ethanol caused a partial dissociation of Gle2p-GFP from the nuclear envelope, and 9% ethanol caused the dissociation of most Gle2p-GFP, as did 10% ethanol. Corresponding to the dissociation of Gle2p-GFP, a partial accumulation of bulk poly(A)+ mRNA in the nucleus was observed with 6% ethanol, and a complete accumulation was seen with 9% ethanol. The minimum concentration of ethanol (6%) causing the accumulation of bulk poly(A)+ mRNA was consistent with the minimum concentration needed to cause the dissociation of Gle2p-GFP. The dissociation of Gle2p from the nuclear envelope correlated well with the blocking of the export of bulk poly(A)+ mRNA. It was reported that Gle2p plays a role in mRNA export in the NPCs (30Murphy R. Watkins J.L. Wente S.R. Mol. Biol. Cell. 1996; 7: 1921-1937Crossref PubMed Scopus (151) Google Scholar). It has been also reported that Gle2p requires physical association with Nup116p to function in vivo (31Bailer S.M. Siniossoglou S. Podtelejnikov A. Helwig A. Mann M. Hurt E. EMBO J. 1998; 17: 1107-1119Crossref PubMed Scopus (117) Google Scholar). Therefore, it seems that the functional activities of Gle2p were decreased by the dissociation from the NPCs under heat-shocked and ethanol-stressed conditions. These results suggest that the dissociation of Gle2p is presumably one of the reasons for the blocking of the export of bulk poly(A)+ mRNA under heat-shocked conditions. Membrane Fluidity Affects the Localization of Gle2p—Next we investigated how the dissociation of Gle2p from the nuclear envelope was caused by heat shock. It is known that heat shock as well as ethanol increases the fluidity of the plasma membrane, followed by changes in the lipid composition of the membranes, including the saturation level and chain length of unsaturated fatty acids (47Alexandre H. Rousseaux I. Charpentier C. FEMS Microbiol. Lett. 1994; 124: 17-22Crossref PubMed Scopus (177) Google Scholar, 48Swan T.M. Watson K. Can. J. Microbiol. 1999; 45: 472-479Crossref PubMed Scopus (69) Google Scholar, 49Örvar B.L. Sangwan V. Omann F. Dhindsa R.S. Plant J. 2000; 23: 785-794Crossref PubMed Google Scholar). There is no information about the fluidity of the nuclear membrane. However, the same effects on the nuclear membrane can be expected for the following reasons: (i) the thermal gradient in each yeast cell seems to be negligible, and (ii) the intracellular and extracellular concentrations of ethanol in yeast cells are comparable, since the cells are permeated by ethanol (50Kunkee R.E. Bisson L.F. Rose A.H. Harrison J.S. The Yeast. 2nd Ed. Academic Press, Inc., New York1993: 39-127Google Scholar). Therefore, we examined whether membrane fluidity affects the localization of Gle2p. Benzyl alcohol (BA), commonly used as a membrane fluidizer, precludes any selective interaction with charged lipid species and fluidizes bilayer membranes (49Örvar B.L. Sangwan V. Omann F. Dhindsa R.S. Plant J. 2000; 23: 785-794Crossref PubMed Google Scholar, 51Zhang G.J. Liu H.W. Yang L. Zhong Y.G. Zheng Y.Z. J. Membrane Biol. 2000; 175: 53-62Crossref PubMed Scopus (37) Google Scholar, 52Sangwan V. Örvar B.L. Beyerly J. Hirt H. Dhindsa R.S. Plant J. 2002; 31: 629-638Crossref PubMed Scopus (293) Google Scholar). We examined whether BA affects the localization of Gle2p in the same manner as heat shock and ethanol. Gle2p-GFP dissociated from the NPCs following the addition of BA (0.8%, v/v) as well as under heat-shocked and ethanol-stressed conditions (Fig. 3). Furthermore, treatment with 0.8% BA for 5 min also blocked the export of bulk poly(A)+ mRNA (Fig. 3). No change in the localization of Rip1p or Nup116p resulted from the treatment with BA (data not shown). We further investigated the concentration-dependent effects of BA on the changes in the localization of Gle2p and the export of bulk poly(A)+ mRNA. As shown in Fig. 3, 0.6% BA caused a partial dissociation of Gle2p-GFP from the nuclear envelope, and 0.7% BA caused the dissociation of more Gle2p-GFP than 0.6% BA. Corresponding to the dissociation of Gle2p-GFP, a partial accumulation of bulk poly(A)+ mRNA in the nucleus was observed with 0.6% BA and an almost complete accumulation with 0.7% BA (Fig. 3). In contrast to the effects of BA, palmitic acid enrichment is known to rigidify membranes. It has been reported that culture in a medium with palmitic acid resulted in a striking increase in the palmitic acid content of phospholipid fatty acids and that such palmitic acid-enriched cells show a decrease in membrane fluidity and an increase in membrane integrity (35Mizoguchi H. Hara S. J. Ferment. Bioeng. 1996; 81: 406-411Crossref Scopus (33) Google Scholar, 36Mizoguchi H. Hara S. J. Ferment. Bioeng. 1997; 83: 12-16Crossref Scopus (24) Google Scholar). We investigated the effects of membrane rigidification on the localization of Gle2p-GFP. The membrane rigidification caused by the enrichment of palmitic acid affected the localization of Gle2p under heat-shocked conditions (Fig. 4), although no effect of the enrichment of palmitic acid was observed under nonstressed conditions (data not shown). Palmitic acid-enriched cells showed a delay in the dissociation of Gle2p after the shift to heat-shocked conditions (Fig. 4). Gle2p-GFP remained around the nuclear envelope in the palmitic acid-enriched cells for a somewhat longer period (more than 15 min) at 42 °C and under 10% ethanol-stressed conditions (Fig. 4), whereas Gle2p-GFP rapidly dissociated in normal cells (within 5 min). In the palmitic acid-enriched cells, the export of bulk poly(A)+ mRNA was carried out even at 42 °C, whereas Gle2p-GFP remained around the nuclea" @default.
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• W2015468911 date "2004-08-01" @default.
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• W2015468911 title "Gle2p Is Essential to Induce Adaptation of the Export of Bulk Poly(A)+ mRNA to Heat Shock in Saccharomyces cerevisiae" @default.
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• W2015468911 doi "https://doi.org/10.1074/jbc.m403692200" @default.
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