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• W2057075953 abstract "Yeast sec mutations define the machinery of vesicular traffic. Surprisingly, many of these mutations also inhibit ribosome biogenesis by reducing transcription of rRNA and genes encoding ribosomal proteins. We observe that these mutants reversibly inhibit protein import into the nucleus, with import cargo accumulating at the nucleoplasmic face of nuclear pore complexes, as when Ran-GTP cannot bind importins. They also rapidly and reversibly relocate multiple nucleolar and nucleoplasmic proteins to the cytoplasm. The import block and relocation are antagonized by overexpression of yeast Ran, Hog1p kinase, or Ssa/Hsp70 proteins or by inhibition of protein synthesis. These nucleocytoplasmic signaling events document an extraordinary plasticity of nuclear organization. Yeast sec mutations define the machinery of vesicular traffic. Surprisingly, many of these mutations also inhibit ribosome biogenesis by reducing transcription of rRNA and genes encoding ribosomal proteins. We observe that these mutants reversibly inhibit protein import into the nucleus, with import cargo accumulating at the nucleoplasmic face of nuclear pore complexes, as when Ran-GTP cannot bind importins. They also rapidly and reversibly relocate multiple nucleolar and nucleoplasmic proteins to the cytoplasm. The import block and relocation are antagonized by overexpression of yeast Ran, Hog1p kinase, or Ssa/Hsp70 proteins or by inhibition of protein synthesis. These nucleocytoplasmic signaling events document an extraordinary plasticity of nuclear organization. endoplasmic reticulum nuclear localization sequence green fluorescent protein protein kinase C heat shock response nuclear pore complex Signaling from the endoplasmic reticulum (ER)1 to the nucleus can result from the proteolytic release of SREB-1 from the cytosolic surface of the ER, which induces lipid biosynthetic enzymes; from the ER “overload” response, which activates NF-κB; from responses to ER calcium depletion, which activate the transcription factors CHOP and YY1; or from the “unfolded protein” response, which induces expression of folding equipment of the ER (1Brown M. Goldstein J. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (3028) Google Scholar, 2Pahl H. Baeuerle P. Trends Cell Biol. 1997; 7: 63-69Abstract Full Text PDF PubMed Scopus (59) Google Scholar, 3Li W. Hsiung Y. Zhou Y. Roy B. Lee A. Mol. Cell. Biol. 1997; 17: 54-60Crossref PubMed Scopus (82) Google Scholar, 4Chapman R. Sidrauski C. Walter P. Annu. Rev. Cell Dev. Biol. 1998; 14: 459-486Crossref PubMed Scopus (205) Google Scholar). In a more general example of signaling from the secretory pathway to the nucleus, interruption of the yeast secretory path inhibits ribosome biogenesis: transcription and processing of rRNA and transcription of genes encoding ribosomal proteins are inhibited (5Liang S. Lacroute F. Kepes F. Eur J. Cell Biol. 1993; 62: 270-281PubMed Google Scholar, 6Mizuta K. Warner J.R. Mol. Cell. Biol. 1994; 14: 2493-2502Crossref PubMed Scopus (126) Google Scholar, 7Li B. Nierras C. Warner J. Mol. Cell. Biol. 1999; 19: 5393-5404Crossref PubMed Scopus (96) Google Scholar, 8Nierras C. Warner J. J. Biol. Chem. 1999; 274: 13235-13241Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Such inhibition is observed with mutants that inhibit any of several steps of membrane traffic. These effects are abrogated by cycloheximide, suggesting that some newly synthesized protein(s) is involved. There is no indication of whether other aspects of nuclear organization may be altered. Strains (TableI) were grown to mid-log phase at 23 °C in appropriate synthetic media and fixed in formaldehyde or shifted to 37 °C for 2 h before fixation. Strains were transformed with pGAL1-SSA1 (CEN4, TRP1; from E. Craig), pGAL-CNR1 (2μ, LEU2; Ref. 17Kadowaki T. Goldfarb D. Spitz L.M. Tartakoff A.M. Ohno M. EMBO J. 1993; 12: 2929-2937Crossref PubMed Scopus (154) Google Scholar), pHOG1 (2μ, TRP1; Ref. 12Brewster J. de Valoir T. Dwer N. Winter E. Gustin M. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1034) Google Scholar), pMPK1-HA(2μ, URA3; Ref. 18Kamada Y. Jung U. Piotrowski J. Levin D. Genes and Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (422) Google Scholar), pHOG1-GFP (CEN, URA3; Ref. 19Ferrigno P. Posas F. Koepp D. Saito H. Silver P. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (352) Google Scholar), pNLS-lacZ (2μ, URA3; Ref.20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar), pMIG1 217–400 -GFP-lacZ (BM3495; 2μ, URA3; Ref. 21deVit R. Waddle J. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (284) Google Scholar), pH2B-lacZ (CEN, URA3; Ref. 22Moreland R. Lengvin G. Singer R. Garcea R. Hereford L. Mol. Cell. Biol. 1987; 7: 4048-4057Crossref PubMed Scopus (136) Google Scholar) by standard procedures and maintained on appropriate dropout plates.Table IYeast strain listStrainRelevant genotypeRef./sourceYPH500Wild type(9Sikorski R. Heiter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar)NY273 act1–2(10Novick P. Botstein D. Cell. 1985; 40: 405-416Abstract Full Text PDF PubMed Scopus (365) Google Scholar)CB101 cdc33–1M. OhnoΔXPO1[pKW457] crm1–1(11Stade K. Ford C. Guthrie C. Weis K. Cell. 1997; 90: 1041-1052Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar)JBY13 Δhog1(12Brewster J. de Valoir T. Dwer N. Winter E. Gustin M. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1034) Google Scholar)MYY260 hsf1(13Smith B. Yaffe M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11091-11094Crossref PubMed Scopus (81) Google Scholar)LUT13 ino1S. HenryWCG4–11a pre1–1S. Jentschprp5–3a prp5–1D. McPheetersPSY876 rsl1–1(14Koepp D. Wong D. Corbett A. Silver P. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar)AFY138 sec1–1A. FranzusoffAFY62 sec7–1A. FranzusoffRSY263 sec12–14R. SchekmanRSY271 sec18–1R. SchekmanRSY281 sec23–1R. SchekmanRSY529 sec62–1R. SchekmanRSY151 sec63–1R. SchekmanV1205 sec1–1 hsf1This studyRSY523 sec61–2 sec18–1R. SchekmanNOY612 srp1–31(15Yano R. Oakes M. Yamaghishi M. Dodd J. Nomura M. Mol. Cell. Biol. 1992; 12: 5640-5650Crossref PubMed Scopus (156) Google Scholar)JN519 ssa1(16Becker J. Walter W. Yan W. Craig E. Mol. Cell. Biol. 1996; 16: 4378-4386Crossref PubMed Scopus (199) Google Scholar)DS2 (S. pombe)Wild typeJ.-A. Wise Open table in a new tab Cells were fixed and processed as described (23Wente S. Rout M. Blobel G. J. Cell Biol. 1992; 119: 705-723Crossref PubMed Scopus (198) Google Scholar). Formaldehyde was added directly to growing cultures for 10 min. Cells were then sedimented and resuspended for 10 min at room temperature in 3.7% formaldehyde, 10% methanol, 0.1 m potassium phosphate, pH 6.5; washed three times in buffer; and washed once in buffered 1.2 msorbitol. They were then carefully spheroplasted, adhered to polylysine-coated slides, dehydrated, and immunostained. For electron microscopy, cells were fixed in 2% formaldehyde, 0.1% glutaraldehyde, spheroplasted, and embedded in LR White. Thin sections were incubated with rabbit anti-β-galactosidase (Cappel, catalog no. 55976), which had been freed from cross-reactivity by preadsorption on fixed yeast, and then with anti-rabbit Ig colloidal gold. Glass bead extracts of cells prepared with buffered SDS were reduced, fractionated by SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose membranes, and probed with the indicated antibodies, using ECL reagents for signal detection. Yeast carrying pGAL-NLS-lacZ were grown in raffinose dropout medium at 23 °C and shifted to the indicated temperatures upon addition of galactose (2% final concentration) 1–2 h before fixation and immunostaining. Yeast carrying pMIG1-GFP-lacZ were grown in glucose dropout medium and then diluted into dropout medium containing 5% glycerol until growth resumed and nuclear GFP could not be detected. (The preliminary growth in glucose medium is needed since growth in glycerol is slow.) To study import, cultures were shifted for 30 min to 23 or 37 °C and then supplemented with glucose (2% final concentration) ± cycloheximide. After 5 min, the cells were quickly washed and examined. Yeast carrying pHOG1-GFP were grown overnight in glucose synthetic medium. At this point, Hog1p-GFP is readily detected in the cytoplasm. The cells were then preincubated for 30 min at 23 or 37 °C and challenged by supplementation with NaCl (0.4 mfinal concentration) for 10 min before examination. To explore the causes of the transcriptional inhibition which is observed in sec mutants, we localized several nuclear proteins insec mutants and observed that they relocate to the cytoplasm. Fig. 1 A, for example, illustrates relocation of the nucleolar prolyl hydroxylase, Fpr3p/Npi46p, at 37 °C upon inhibition of vesicle exocytosis insec1–1, upon inhibition of transport through the Golgi insec7–1, and upon inhibition of several steps of membrane traffic in sec18–1. Comparable relocation is also seen insec12–4 and sec23–1, which block export from the ER. Several other nucleolar proteins also relocate under these conditions (Cbf5p, Nop1p, Nop4p, Nsr1p, and Ssb1p), as does the nucleoplasmic protein, Npl3p/Mtr13p (20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar, 24Woolford Jr., J.L. Adv. Genet. 1991; 29: 63-118Crossref PubMed Scopus (63) Google Scholar, 25Jordan E. Shaw P. Annu. Rev. Cell Biol. 1995; 11: 93-122Crossref Scopus (409) Google Scholar, 26Warner J. Microb. Rev. 1996; 53: 256-271Crossref Google Scholar). A substantially weaker effect is seen in sec63–1, which interrupts translocation into the ER. To interrupt membrane traffic in the absence of mutant proteins and temperature increase, we have used brefeldin A (27Turi T. Mueller U. Sazer S. Rose J. J. Biol. Chem. 1996; 271: 9166-9171Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) and also observe distinct although less extreme relocalization of a nucleolar protein within 30 min at 30 °C (Fig.1 B). The relocation in sec mutants of Fpr3p and the yeast fibrillarin homologue, Nop1p, is strikingly inhibited by cycloheximide (Fig. 1 A) or by the transcriptional inhibitor, thiolutin (data not shown). At least Fpr3p is known not to shuttle (20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar). Moreover, relocation of Fpr3p and Nop1p is reversible: after incubatingsec1–1, sec7–1, or sec18–1 for 2 h at 37 °C, return to 23 °C for 2 h ± 100 μg/ml cycloheximide leads to reconcentration of these antigens in the nucleolus, unless ATP synthesis is inhibited. This evidence of reversibility demonstrates the significant stability of at least Fpr3p and Nop1p, thereby ruling out an alternate explanation of their appearance in the cytoplasm: ongoing synthesis and lack of import. Relocation of Fpr3p and Nop1p is not seen in wild type over 1 h at 30–37 °C ± cycloheximide or thiolutin (23Wente S. Rout M. Blobel G. J. Cell Biol. 1992; 119: 705-723Crossref PubMed Scopus (198) Google Scholar), or in several other temperature-sensitive mutants, e.g. act1–2, cdc33–1, pre1–1, andprp5–1, as well as in sec62–1, which inhibits protein translocation into the ER (data not shown). Our earlier studies have documented a close relation between relocation of nucleolar proteins to the cytoplasm and inhibition of nuclear import (20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar). We have therefore inquired whether the three secmutants inhibit import of NLS-β-galactosidase, which has the nuclear localization signal (NLS) of SV40 large T antigen, and other proteins that require distinct import equipment: Mig1p-GFP-β-galactosidase, H2B-GFP, and Hog1p-GFP (19Ferrigno P. Posas F. Koepp D. Saito H. Silver P. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (352) Google Scholar, 21deVit R. Waddle J. Johnston M. Mol. Biol. Cell. 1997; 8: 1603-1618Crossref PubMed Scopus (284) Google Scholar, 22Moreland R. Lengvin G. Singer R. Garcea R. Hereford L. Mol. Cell. Biol. 1987; 7: 4048-4057Crossref PubMed Scopus (136) Google Scholar). Import of each of these proteins can be readily documented in wild type at 23 °C or 37 °C, but is strongly inhibited in the sec mutants (Fig.2 A). When NLS-β-galactosidase is induced for 1 or 2 h at 37 °C, it is detected at the periphery of the nucleus and in the cytoplasm, with the peripheral distribution being most visible in sec1–1 andsec7–1 and the cytoplasmic signal most visible insec18–1 (Fig. 2 A). Fig. 2 (B andC) shows that a discrete amount of the import cargo actually enters the extreme periphery of the nucleoplasm. This is quite different from the docking of cargo at cytosolic NPC-associated sites in the absence of an energy source (28Richardson W. Mills A. Dilworth S. Laskey R. Cell. 1988; 52: 655-666Abstract Full Text PDF PubMed Scopus (374) Google Scholar). Thus, the secmutants may inhibit release of import cargo from the nucleoplasmic face of the NPC, as when the import factor, importin β, cannot interact with the GTP-bound nuclear pool of the GTPase, Ran, which governs nucleocytoplasmic transport (29Gönlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15 (in press)Google Scholar, 30Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (535) Google Scholar). As for relocation of nucleolar proteins, protein synthesis is required for inhibition of import; for example, Mig1p-GFP-β-galactosidase normally enters the nucleus upon shift from raffinose to glucose medium and this entry is inhibited in the sec mutants at 37 °C unlesscycloheximide is present (data not shown). Since Ran cycle mutants inhibit nuclear import and since the site of inhibition of import in secmutants suggests that there is a defect at the level of the Ran cycle, we have inquired whether the Ran-GAP mutant, rna1–1, and the Ran-GEF mutant, mtr1–1, relocate nucleolar proteins. As shown in Fig. 1 C, this is the case. Moreover, overexpression of yeast Ran strikingly protects import and reduces the relocation of Fpr3p and Nop1p in sec1–1, sec7–1, and sec18–1(Figs. 1 A, 2 C, and 3 B). Although these observations are consistent with the notion that import is critical for retention of nuclear proteins, inhibition of the importin α/β import path in corresponding mutants does not cause relocation ((srp1–31, rsl1–1) (Refs. 14Koepp D. Wong D. Corbett A. Silver P. J. Cell Biol. 1996; 133: 1163-1176Crossref PubMed Scopus (114) Google Scholar, 15Yano R. Oakes M. Yamaghishi M. Dodd J. Nomura M. Mol. Cell. Biol. 1992; 12: 5640-5650Crossref PubMed Scopus (156) Google Scholar and29Gönlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15 (in press)Google Scholar, 31Corbett A. Silver P. Microbiol. Mol. Biol. Rev. 1997; 61: 193-210Crossref PubMed Scopus (170) Google Scholar; data not shown). Furthermore, Ran does remain concentrated in the nucleus as judged by immunofluorescence in the secmutants (data not shown) and, although more subtle modifications may occur, the abundance and 1D gel mobility of yeast Ran, Rna1p and Mtr1p/Prp20p do not change over 2 h at 37 °C (Fig.3 A). Some aspects of cell physiology in the sec mutants (increased concentration of cytosolic proteins, limitation of the cell perimeter, altered relation between the cell wall and plasma membrane, etc.) may resemble the effects of exposure to media of altered tonicity. We have therefore asked whether expression from a high copy plasmid of the key MAP kinases implicated in resistance to hypertonic and hypotonic stress (Hog1p or Mpk1p (the terminal kinases of the PKC signaling path)), ensures protein import and nucleolar coherence in thesec mutants at 37 °C (12Brewster J. de Valoir T. Dwer N. Winter E. Gustin M. Science. 1993; 259: 1760-1763Crossref PubMed Scopus (1034) Google Scholar, 32Posas F. Saito H. Science. 1997; 276: 1702-1705Crossref PubMed Scopus (472) Google Scholar). Remarkably, studies of the import of Mig1p-GFP-β-galactosidase and localization of Fpr3p or Nop1p show that Hog1p does have these effects (Figs. 1 A and2 C). Mpk1p does not provide protection (data not shown). Activation of Hog1p by hypertonic stress is accompanied by its phosphorylation and transient entry into the nucleus, its exit being mediated by the importin β family member, Crm1p (19Ferrigno P. Posas F. Koepp D. Saito H. Silver P. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (352) Google Scholar, 33Reiser V. Ruis H. Ammerer G. Mol. Biol. Cell. 1999; 10: 1147-1161Crossref PubMed Scopus (178) Google Scholar). Since the protection by Hog1p may also reflect its entry into the nucleus, we have monitored the distribution of Hog1p-GFP in sec mutants. We observe that it remains concentrated in the cytoplasm, and that this is true even upon hypertonic shock (data not shown). Moreover, experiments in which Hog1p-GFP is expressed in crm1–1 show that Hog1p-GFP cannot be trapped in the nucleus, i.e. it does not cycle through the nucleus under normal growth conditions. Any relevant targets of Hog1p kinase activity therefore may be external to the NPC. Nevertheless, upon overexpression there may be some increased nuclear titer of Hog1p. Interestingly, HOG1 is not essential, and a Δhog1 strain is not obviously deficient in nuclear import or nucleolar organization. There has been only limited investigation of the heat shock response (HSR) in secmutants (34Normington M. Kohno K. Kozutsumi Y. Gething M.-J. Sambrook J. Cell. 1989; 57: 1223-1236Abstract Full Text PDF PubMed Scopus (305) Google Scholar, 35Rose M. Misra L. Vogel J. Cell. 1989; 57: 1211-1221Abstract Full Text PDF PubMed Scopus (528) Google Scholar). We observe that when sec1–1, sec7–1 orsec18–1 is incubated at 37 °C, there is a strong and sustained HSR, as judged by transcription from a reporter plasmid, pHSE 2- lacZ. The HSR exceeds that seen in wild type cells, both in intensity and in duration, withsec18–1 being the strongest. Moreover, immunoblotting and pulse labeling of newly synthesized proteins show clear induction of Hsp104 and Hsp70 proteins in these mutants at 37 °C. Nevertheless, by comparison to several temperative-sensitive strains that do not affect membrane traffic or relocate nuclear proteins (act1–2, cdc33–1, pre1–1, prp5–1, sec62–1), the HSR insec mutants is of intermediate intensity. In fact,sec62–1 induces a HSR that is substantially stronger than all the other strains. Moreover, incubation of wild type at 37 °C or treatment with doses of ethanol, arsenite, and dihydrosphingosine known to produce a HSR does not cause relocation. Thus, the HSR does not parallel relocation. Interestingly, inhibition of import of Mig1p-GFP-β-galactosidase and relocation of the nucleolar proteins do not require induction of heat shock proteins that depend on heat shock transcription factor (Hsf1p), judging from examination of asec1–1 hsf1 double mutant. Moreover, the hsf1mutant (and ssa1) does not itself inhibit import or cause relocation (data not shown). Although a HSR does not necessarily perturb the nucleus and although Hsf1p function is not needed to cause such perturbation, the impact ofsec mutations on the nucleus appears similar to the effects of incubation at elevated temperatures. Incubation at 42 °C, for example, also inhibits protein import and causes nucleolar proteins to relocate to the cytoplasm (20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar), and incubation even at 37 °C selectively inhibits transcription of ribosomal protein genes (26Warner J. Microb. Rev. 1996; 53: 256-271Crossref Google Scholar, 37Lashkari D. DeRisi J. McCusker J. Namath A. Gentile C. Hwang S. Brown P. Davis R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13057-13062Crossref PubMed Scopus (532) Google Scholar). Since Ssa/Hsp70 proteins are needed to protect yeast against relocation at elevated temperature (20Liu Y. Liang S. Tartakoff A. EMBO J. 1996; 15: 6750-6757Crossref PubMed Scopus (46) Google Scholar), we have inquired whether overexpression of Ssa1p at 37 °C can protect the sec mutants against nuclear changes. Strikingly, Ssa1p overexpression does protect against inhibition of import and relocation (Figs. 1 A and2 C). Expression of yeast Ran or Hog1p does not alter levels of Ssa proteins, as detected by Western blotting (data not shown). Thus, the protection due to Ran or Hog1p appears not to be mediated by increasing the amount of Ssa proteins. The surprising rapid and reversible nuclear phenotypes of sec mutants are reminiscent of several previous, disparate reports, in addition to the inhibition of ribosome biogenesis mentioned in the Introduction. For example, there are unpublished observations of lack of import of a ribosomal protein-β-galactosidase fusion in a sec23 mutant (cited in Ref. 38Hicke L. Schekman R. EMBO J. 1989; 8: 1677-1684Crossref PubMed Scopus (74) Google Scholar). Moreover, although we find that sec63 mutants do not have a strong phenotype, yeast carrying point mutations in Sec63p mislocalize an unidentified nucleolar protein and a newly synthesized SV40 large T antigen-NLS-invertase fusion to the cytoplasm (39Nelson M. Kurihara T. Silver P. Genetics. 1993; 134: 159-173Crossref PubMed Google Scholar). Strikingly, it has also been reported that Schizosaccharomyces pombe can be rendered resistant to brefeldin A by overexpression of a homologue of a Ran-binding protein or by mutating the exportin, Crm1p (27Turi T. Mueller U. Sazer S. Rose J. J. Biol. Chem. 1996; 271: 9166-9171Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). We suggest thatsec1, sec7, and sec18 each send equivalent signals, which impact on Ran cycle function and cause relocation of nuclear proteins. We do not observe equivalent perturbation of the nucleus in sec mutants that block polypeptide translocation into the ER; however, this may be because of the major HSR that such mutants elicit. Why does interruption of membrane traffic perturb the nucleus? We can exclude several possibilities. (a) The mere presence of mutant Sec proteins might perturb the nucleus; however, neither haploid sec18–1 carrying aSEC18 plasmid nor sec18–1/SEC18 heterozygous diploids relocate nucleolar proteins at 37 °C (data not shown). (b) To learn whether relocation results from overaccumulation of newly synthesized proteins or lipids upstream from the secretion blocks, we have inquired: 1) whether relocation is seen in a sec61–2 sec18–1 double mutant, sincesec61–2 reduces translocation of newly synthesized proteins into the ER at the restrictive temperature (41Deshaies R. Schekman R. J. Cell Biol. 1989; 109: 2653-2664Crossref PubMed Scopus (133) Google Scholar); and 2) whether relocation can be eliminated by inhibitors of fatty acid and sterol biosynthesis (cerulenin, zaragozic acid; Ref. 42Schneiter R. Hitomi M. Ivessa A.S. Kohlwein S.D. Tartakoff A.M. Mol. Cell. Biol. 1996; 16: 7161-7172Crossref PubMed Scopus (153) Google Scholar). These experiments do not support the hypothesis that overaccumulation in the ER causes relocation. Relocation in sec18–1 sec61–2 is comparable tosec18–1 and the inhibitors of lipid synthesis (which themselves do not affect the distribution of nucleolar proteins in wild type cells), do not protect the sec mutants against the impact of the 37 °C incubation (data not shown). (c) An unfolded protein response is induced by tunicamycin and tunicamycin does inhibit ribosome biogenesis (6Mizuta K. Warner J.R. Mol. Cell. Biol. 1994; 14: 2493-2502Crossref PubMed Scopus (126) Google Scholar, 8Nierras C. Warner J. J. Biol. Chem. 1999; 274: 13235-13241Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), but tunicamycin treatment does not cause relocation of nucleolar proteins over 2 h at 30 °C (data not shown). (d) Cycloheximide or transcriptional inhibitors do not cause relocation of nucleolar proteins. Moreover, these inhibitors protect the nucleus of sec mutants. It therefore cannot be true that mere interruption of delivery of one or more newly synthesized proteins to the cell surface is sufficient to perturb the nucleus. (e) “Unbalanced growth” may occur when thesec mutants continue to increase their mass but are unable to enlarge their surface area. A comparable imbalance has been suggested to underlie the “inositol-less death” that occurs when inositol auxotrophs are deprived of inositol, unless cycloheximide is present (43Culbertson M. Henry S. Genetic s. 1975; 80: 23-40Crossref Google Scholar). Nevertheless, inositol withdrawal from an ino1strain does not relocate nucleolar proteins over 4 h at 30 °C (data not shown). As a conservative hypothesis, since overexpression either Ssa1p or yeast Ran protects the nucleus, we propose that interruption of membrane traffic sequesters or inactivates Ssa proteins, which are known to be critical for import (45Shulga N. Roberts P. Gu Z. Spitz L. Tabb M. Nomura M. Goldfarb D. J. Cell Biol. 1996; 135: 329-339Crossref PubMed Scopus (186) Google Scholar, 46Lamian V. Small G. Feldherr C. Exp. Cell Res. 1996; 228: 84-91Crossref PubMed Scopus (35) Google Scholar) (Fig.4). Evidence consistent with there being a reduction of levels of free Ssa proteins in sec mutants is provided by the induction of transcription from the HSR reporter plasmid, considering that reduction of free Hsp70/Ssa protein levels is known to induce a HSR (44Craig E. Gross C. Trends Biochem. Sci. 1991; 16: 135-140Abstract Full Text PDF PubMed Scopus (479) Google Scholar). 2We have also obtained evidence of a functional interaction between Ssa1p and the Ran cycle; overexpression of Ssa1p suppresses the protein relocalization seen in Ran-GEF mutants (mtr1/prp20), and the allele specificity of this suppression is the same as for Cnr1p (yeast Ran) suppression ofmtr1/prp20 mutants (19Ferrigno P. Posas F. Koepp D. Saito H. Silver P. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (352) Google Scholar). This is consistent with biochemical observations of association between these proteins in animal cells. Several previous studies have also implicated Hsp70 proteins in nuclear import. Despite these indications of Ssa protein involvement, mere loss of Ssa function in ssa1 and hsf1 strains does not inhibit import or relocate nuclear proteins at 37 °C. We therefore propose that interruption of membrane traffic must also cause other events that are functionally linked to components of the Ran cycle,e.g. phosphorylation of critical substrates. Judging from our observations of protection by Hog1p and the recent report that protein kinase C (but not Hog1p or cAMP-dependent protein kinase) is required forsec perturbation of the nucleus (8Nierras C. Warner J. J. Biol. Chem. 1999; 274: 13235-13241Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), Hog1p and PKC paths play opposing roles, possibly acting on the same targets. We suggest that subsequent loss of Ran cycle function then inhibits protein import and that inhibition of import leads to relocation of nuclear proteins and therefore inhibits transcription. Once relocation of nuclear proteins begins, if key factors that are needed for import are also perturbed, the process may accelerate. The cycloheximide sensitivity of nuclear perturbation by secmutants may reflect a need for ongoing synthesis of some key protein (s), e.g. a protein that activates PKC. Since Hsp70 proteins participate in conformational maturation of newly synthesized proteins (47Bush G. Meyer D. J. Cell Biol. 1996; 135: 1229-1237Crossref PubMed Scopus (35) Google Scholar, 48Eggers D. Welch W. Hansen W. Mol. Biol. Cell. 1997; 8: 1559-1573Crossref PubMed Scopus (86) Google Scholar, 49Pfund C. Lopez-Hoyo N. Ziegelhofer T. Schilke B. Lopez-Buesa P. Walter W. Wiedmann M. Craig E. EMBO J. 1998; 17: 3981-3989Crossref PubMed Scopus (190) Google Scholar), an alternate possibility is that Hsp70 proteins are most available for import when protein synthesis is inhibited. Thus, in the presence of cycloheximide, although additional Ssa proteins are not synthesized, ambient levels may remain sufficient to protect the nucleus. There may also be a specific molecular link between vesicular transport and nucleocytoplasmic transport. For example, both Sec13p and one ER membrane protein interact with nucleoporins (50Siniossoglou S. Wimmer C. Rieger M. Doye V. Tekotte H. Weise C. Emig S. Segref A. Hurt E. Cell. 1996; 84: 265-275Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 51Ho A. Raczniak G. Ives E. Wente S. Mol. Cell. Biol. 1998; 9: 355-373Crossref Scopus (35) Google Scholar), a member of the importin β superfamily is implicated in both protein secretion and nuclear transport (52Chow T. Ash J. Dignard D. Thomas D. J. Cell Sci. 1992; 101: 709-719Crossref PubMed Google Scholar, 53Rout M. Blobel G. Aitchison J. Cell. 1997; 89: 715-725Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), and mutations in splicing factors can inhibit vesicular transport (54Chen E. Frand A. Chitouras E. Kaiser C. Mol. Cell. Biol. 1998; 18: 7139-7146Crossref PubMed Scopus (24) Google Scholar). Since GTPases of the Ras superfamily are critical for both vesicular transport and nuclear transport (55Novick P. Zerial M. Curr. Opin. Cell Biol. 1997; 9: 496-504Crossref PubMed Scopus (666) Google Scholar,56Nimnual A. Yatsula B. Bar-Sagi D. Science. 1998; 279: 560-563Crossref PubMed Scopus (389) Google Scholar), it is also of interest that proteins have been identified which interact with both Ran and Rabs (57Vithalani K. Parent C. Thorn E. Penn M. Larochelle D. Devreotes P. de Lozanne A. Mol. Biol. Cell. 1998; 8: 3095-3106Crossref Scopus (30) Google Scholar). Moreover, at least one Rab protein and Hsp70 interact with stress signaling paths (58Ren M. Zeng J. Lemos-Chiarandini C. Rosenfeld M. Adesnik M. Sabatini D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5151-5155Crossref PubMed Scopus (88) Google Scholar, 59Gabai V. Meriin A. Mosser D. Caron A. Rits S. Shifrin V. Sherman M. J. Biol. Chem. 1997; 272: 18033-18037Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). There are other circumstances in which nucleocytoplasmic transport is perturbed or nuclear proteins relocate to the cytoplasm, perhaps because of inhibition of protein import. Thus, several viruses that replicate in the cytoplasm of animal cells relocate specific nuclear proteins to the cytoplasm and the M protein of vesicular stomatitis virus inhibits nucleocytoplasmic transport, which depends on Ran (60Her L.-S. Lund E. Dahlberg J. Science. 1997; 276: 1845-1848Crossref PubMed Scopus (155) Google Scholar). Although many nuclear proteins appear not to shuttle under normal growth conditions, they may do so under extreme circumstances. We thank P. Belhumeur, J. Brodsky, M. Caizergas-Ferrer, E. Craig, R. DeVit, D. Drubin, S. Emr, M. Ferrer, A. Franzusoff, D. Goldfarb, M. Gustin, A. Hopper, S. Jentsch, S. Lemmon, S. Lindquist, M. Nomura, P. Novick, G. Payne, H. Pelham, R. Schekman, P. Silver, J. Thorner, J.-A. Wise, and M. Yaffe for yeast strains, antibodies, and plasmids and Drs. M. Snider and J.-A. Wise for comments on the text." @default.
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• W2057075953 title "An Unexpected Link between the Secretory Path and the Organization of the Nucleus" @default.
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