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Genetics, Vol. 168, 1899-1913, December 2004, Copyright © 2004
doi:10.1534/genetics.104.032961
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,1
* Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853
1 Corresponding author: College of Agriculture and Life Sciences, Cornell University, 260 Roberts Hall, Ithaca, NY 14853.
E-mail: sah42{at}cornell.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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and ire1, are also inositol auxotrophs, a phenotype associated with defects in expression of INO1, the most highly regulated of a set of genes encoding enzymes of phospholipid metabolism. We now demonstrate that the UPR plays a functional role in membrane trafficking under conditions of secretory stress in yeast. Mutations conferring a wide range of membrane trafficking defects exhibited negative genetic interaction when combined with ire1 and hac1. At semipermissive temperatures, carboxypeptidase Y transit time to the vacuole was slower in Sec– cells containing an ire1 or hac1 mutation than in Sec– cells with an intact UPR. The UPR was induced in Sec– cells defective in subcellular membrane trafficking events ranging from ER vesicle trafficking to distal secretion and in erg6 cells challenged with brefeldin A. However, the high levels of UPR induction observed under these conditions were not correlated with elevated INO1 expression. Indeed, many of the Sec– mutants that had elevated UPR expression at semipermissive growth temperatures failed to achieve wild-type levels of INO1 expression under these same conditions.
Cells carrying ire1, hac1, or rlg1-100 mutations are sensitive to drugs such as tunicamycin, which causes accumulation of misfolded proteins in the ER (NIKAWA et al. 1996; SHAMU and WALTER 1996; SIDRAUSKI et al. 1996; COX et al. 1997). In addition, ire1, hac1, or rlg1-100 mutants are inositol auxotrophs (NIKAWA and YAMASHITA 1992; COX et al. 1993; NIKAWA et al. 1996; SIDRAUSKI et al. 1996), a phenotype associated with defects in expression of genes related to phospholipid metabolism, especially INO1, the structural gene encoding myo-inositol 3-phosphate synthase (for review see HENRY and PATTON-VOGT 1998). INO1 and coregulated genes of phospholipid metabolism contain the inositol-sensitive upstream activating sequence (UASINO) repeated element in their promoters and exhibit complex transcriptional regulation in response to a variety of environmental factors including the availability of soluble precursors of phospholipid metabolism such as inositol (CARMAN and HENRY 1999). Wild-type yeast cells express INO1 and other UASINO-containing genes at a high level when inositol is limiting in the growth medium and repress these same genes when inositol is plentiful (HIRSCH and HENRY 1986; GREENBERG and LOPES 1996; CARMAN and HENRY 1999; LOEWEN et al. 2004). Cox et al. (1997) reported that the UPR is activated in the absence of inositol and suggested that the activation of the UPR might be directly involved in the mechanism by which INO1 transcription is activated when inositol is limiting.
In an earlier study, we explored the relationship between UPR induction and INO1 expression in response to signals generated due to altered phospholipid metabolism in mutants defective in the phosphatidylinositol (PI)/phosphatidylcholine (PC) transporter encoded by the SEC14 gene (CHANG et al. 2002). While the SEC14 gene product is essential for viability, the growth and secretory defects of sec14 mutants can be suppressed by mutations in the cytidine 5'-diphosphate (CDP) choline pathway for PC biosynthesis, such as cki1, pct1, and cpt1 (CLEVES et al. 1991). Double mutants, such as sec14ts cki1, when shifted to a semipermissive or restrictive temperature for sec14ts, exhibit multiple abnormalities in phospholipid metabolism, including both elevated PC turnover via a phospholipase D catalyzed route and elevated expression of INO1 (PATTON-VOGT et al. 1997; SREENIVAS et al. 1998). Under these same conditions sec14ts cki1 cells also exhibit high levels of UPR activation (CHANG et al. 2002). These observations are consistent with the hypothesis of Cox et al. (1997) that the UPR and inositol responses are linked and are branches of the same signaling pathway. However, when the UPR mutations ire1 and hac1 were crossed into the sec14ts cki1 background, elevated INO1 expression was still observed at the sec14ts semipermissive temperature, indicating that UPR activation is not required or responsible for the elevated INO1 expression observed in sec14ts cki1 cells (CHANG et al. 2002).
The effects we observed in sec14ts cki1 cells led us to question whether defects in other steps in the secretory pathway might also activate the UPR and/or influence INO1 expression and, thus, provide further insights into the role of the UPR in the secretory pathway and in INO1 regulation. In this study, we have examined the role of the UPR in membrane trafficking and in INO1 expression under conditions of secretory stress induced by lesions in a number of transport steps of the secretory pathway. We report that cells defective in a wide range in membrane trafficking steps exhibit UPR activation and that a functional UPR plays a role in the survival of cells with inefficient membrane trafficking due to a wide range of secretory defects. Thus, the UPR appears to be essential to growth under conditions in which secretory capacity is diminished. However, Sec– mutants grown under the conditions that result in UPR activation exhibit reduced, rather than elevated, INO1 expression. Thus, the relative levels of UPR activation and INO1 transcription are not correlated under conditions of secretory stress.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Yeast strains:
The genotypes and sources of strains are listed in Table 1. Yeast strains listed in Table 1 are of the S288C and W303 genetic backgrounds. Strains were constructed by standard tetrad analysis (SHERMAN et al. 1978; ROSE et al. 1990). Briefly, HCY104, HCY105, HCY106, HCY107, HCY126, and HCY362 were obtained as meiotic spore colonies from diploid deletion strains obtained from the Research Genetics (Huntsville, AL) strain collection. To obtain an ire1 strain isogenic with the genetic background of Sec– strains used in this study, the HCY405 strain was constructed using PCR-based gene disruption of IRE1 in the CKY8 genetic background. A 1.5-kb deletion cassette containing the bacterial KanMX gene was amplified by PCR using pFA6-KanMX4 as a template and IRE1-S1 and IRE1-S2 as the primers (WACH et al. 1994). The KanMX4-containing PCR fragment was included at each end, 45 bp (85–129) and 43 bp (3039–3081), of the IRE1 ORF, resulting in replacement of the IRE1 gene with KanMX. The 1.5-kb deletion cassette was transformed into CKY8 using the lithium acetate (LiAc) method as described previously (HILL et al. 1991). Transformants were selected on YEPD containing 200 mg/liter geneticin (YEPD + G418; Calbiochem, La Jolla, CA). To verify the correct ORF replacement, the size of the insert (1.5 kb) was checked by PCR using a set of primers flanking the disrupted gene (IRE1-S3 and IRE1-S4) and genomic DNA isolated from each of the putative deletion mutants as a template. HCY423 was obtained by mating HCY405 and CKY46. To obtain a hac1 mutant in the same genetic background, a hac1 meiotic spore generated from the heterozygous diploid strain collection (Research Genetics) was backcrossed to CKY49 to generate HCY126.
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or Sec– hac1, were constructed by crossing the various ire1 or hac1 strains to the various Sec– strains. HCY280 and HCY283 were obtained by mating HCY423 and RSY263. HCY427 and HCY428 were obtained by mating HCY423 and RSY267. HCY425 and HCY422 were obtained by mating HCY405 and CKY46. HCY437 and HCY435 were obtained by mating HCY423 and RSY269. HCY442 and HCY444 were obtained from the mating between HCY423 and RSY271. HCY455 and HCY457 were obtained from the cross of HCY423 and RCY279. HCY448 and HCY446 were obtained by mating HCY423 and RCY281. HCY470 and HCY473 were obtained by mating HCY405 and JBY318. HCY178, HCY179, HCY180, and HCY181 were obtained from the cross of HCY126 and CKY49. HCY462, HCY463, HCY465, and HCY464 were the products of a cross between HCY362 and HCY401. HCY495 and HCY496 were obtained from a mating between HCY107 and RCY243. Two sets of four spores containing the tetratype genotypes derived from a single tetrad (HCY462–HCY465 and HCY466–HCY469) were generated by crossing HCY362 (erg6) to HCY401 (ire1) and HCY030 (hac1), respectively.
Transformation with pHAC1 and pHAC1i:
Various Sec– strains were transformed with the HAC1- and HAC1i-containing plasmids (pHAC1 and pHAC1i, CEN/LEU2 marker; gifts from Dr. Kazutoshi Mori, Kyoto University, Kyoto, Japan). The sec13 strain, CKY480 (see Table 1 for the full genotype), carrying the wild-type SEC13 gene on a plasmid (pRS316; CEN; URA3 marker; provided by Dr. Chris Kaiser) was transformed with pHAC1i or an empty vector (YCp50; CEN; LEU2 marker). Since the SEC13 gene is essential for growth, the SEC13 null mutant is obligated to carry and express the wild-type SEC13 gene on a plasmid for its survival. The double transformants simultaneously carrying pSEC13 and an empty vector or pSEC13 and pHAC1i were grown for 1 day on plates containing synthetic medium lacking uracil and leucine and then replica plated onto plates lacking leucine and containing 5-fluoroorotic-acid (5-FOA) and incubated for an additional 3 days at room temperature. The expression of the URA3 gene is toxic to cells exposed to 5-FOA, forcing the transformants to lose the pSEC13 plasmid, thus obligating them to depend on expression of pHAC1i for survival.
ß-Galactosidase assays:
To assay UPRE-CYC-lacZ expression, strains were transformed to uracil prototrophy with pJC104, containing UPRE-CYC-lacZ, provided by Dr. Peter Walter (COX and WALTER 1996). Transformants were precultured to midlogarithmic phase of growth at 25° in synthetic medium lacking uracil. Cells were collected by centrifugation, washed with sterile dH2O, and diluted to OD600 = 0.1 in medium lacking uracil and shifted to the designated temperature to grow for 3 additional hours. Samples (1 ml) were taken and analyzed for ß-galactosidase activity using the Yeast ß-galactosidase assay kit (Pierce, Rockford, IL). For UPRE-CYC-lacZ expression assay in BFA-treated erg6 cells, HCY104 and HCY362 were transformed with pJC104 (UPRE-CYC-lacZ). Cells were prepared as described above except that their initial OD600 was 0.5. Indicated amounts of BFA were added to medium lacking uracil and cells were grown at 30°, and ß-galactosidase activity was measured as described above.
To assess INO1 expression, strains were transformed to uracil prototrophy with pJH359 (INO1-CYC-lacZ) and transformants were precultured at 25° to midlog phase of growth in synthetic medium with (I+) or without (I–) inositol. Cells were then shifted to the indicated temperature for 3 hr and ß-galactosidase was measured.
Northern blot analysis:
Cells were precultured overnight to midlogarithmic phase of growth at 25° in YEPD medium. Wild-type cells were shifted to YEPD medium at 30° in the presence or absence of 1 mM tunicamycin. Sec– strains were shifted to YEPD medium at their semipermissive temperatures. Isolation of RNA was performed by hot phenol extraction. Northern analysis was performed by running 10-µg samples of RNA loaded onto 1% agarose, 6% formaldehyde, 1x MOPS gels and capillary transferred to Nylon Plus (QIAGEN, Valencia, CA) membrane. Prehybridization and hybridization conditions were as described in HIRSCH and HENRY (1986). Hybridization probes for ACT1 (MARYKWAS and FOX 1989) and HAC1 were prepared using a riboprobe in vitro transcription system. ACT1 was linearized with BamHI and riboprobe was synthesized with SP6 RNA polymerase according to manufacturer's instructions (Promega, Madison, WI). The 449-bp SalI fragment from YCp-HAC1 was subcloned into pGEM1 (Promega) through the SalI site (pGEM-HAC1). pGEM-HAC1 was linearized with HindIII. Riboprobe was synthesized with T7 RNA polymerase according to manufacturer's instructions.
Spheroplast pulse-chase labeling and immunoprecipitation:
Spheroplast labeling and immunoprecipitation were carried out essentially as described by WEBB et al. (1997). All strains were grown at the temperatures indicated in Figure 5 and in the text to midlogarithmic phase (OD600 = 0.6–1.0) at 24° in either Wickerman's sulfate-free medium containing 200 µM MgSO4 and 0.2% yeast extract (wild type, sec13ts-1, and sec13ts-1 ire1 strains) or synthetic complete medium lacking leucine (sec13ts-4 hac1 pHAC1i transformants). Five OD600 units of cells per time point were collected and converted to spheroplasts in Wickerham's medium containing 1 M sorbitol and 1 mg/ml bovine serum albumin by digestion with 30 units/OD600 lyticase (Sigma) at room temperature for 45 min. Spheroplasts from wild-type, sec13ts-1, and sec13ts-1 ire1 strains were preincubated for 15 min at 30°; pulse labeled with 50 µCi Trans-35S labeling reagent (ICN Radiochemicals, Irvine, CA) in spheroplasting medium for 5 min at 30°; and chased by adding 5 mM methionine and 1 mM cysteine, 0.2% yeast extract, and 2% glucose and incubating for 0, 10, 20, or 30 min at 30°. Pulse-chase labeling of spheroplasts from sec13ts-4 hac1 pHAC1i transformants was carried out as described above except incubations were performed at 35°. Protein extracts were prepared by trichloracetic acid precipitation of cell pellets followed by resuspension and boiling with glass beads in suspension buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1% SDS). After a fivefold dilution with IP dilution buffer (60 mM Tris-HCl pH 7.5, 6 mM EDTA, 190 mM NaCl, 1.25% Triton X-100), samples were precleared by centrifugation to remove insoluble material. Carboxypeptidase Y (CPY) was immunoprecipitated from each sample by incubating with rabbit anti-CPY antibodies (Rockland, Gilbertsville, PA) and protein A-sepharose CL-4B (Sigma) in IP buffer (50 mM Tris-HCl pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 0.2% SDS). Washed immunoprecipitates were eluted with sample buffer, separated on 8% SDS polyacrylamide gels, and visualized by autoradiography. A digital image of the gel was acquired by scanning the autoradiograph. To obtain the signal intensity of each species of CPY, the digital image was analyzed by Kodak digital science 1D image analysis software (Eastman Kodak, Rochester, NY). The relative total labeling of CPY was estimated by summing up the signal intensity of p1, p2, and mature (mCPY) on each autoradiograph. The proportion of each species of was then calculated by dividing the separate signal intensity of p1, p2, or m by the sum of the intensity of the three bands.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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(BECHERER et al. 1996), were also analyzed). Activation of the UPR in wild-type and mutant strains (Figure 1A) was assayed using an UPRE-CYC-lacZ reporter gene, as described in MATERIALS AND METHODS. Sec– strains were assayed only under conditions at which they could grow continuously. Therefore, ß-galactosidase activity is reported for only a subset of strains at 33° (Figure 1A). Wild-type cells exhibited the expected low levels of UPRE expression at 25° and 30°, and only a slight increase in UPRE expression was observed at 33° (Figure 1A). In contrast, virtually all of the temperature-sensitive Sec– mutants showed elevated UPRE activation compared to wild type when grown at their semipermissive temperatures of 30° or 33° (Figure 1A).
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The sec17ts-1, sec18ts-1, and sec22ts-3 strains, which are defective in vesicle docking and fusion, also exhibited a five- to eightfold elevation in UPRE-driven ß-galactosidase activity at 30° (Figure 1A). In the sec21ts-1 strain, which has a defect in COPI coatomer, UPRE expression was elevated more than fivefold at 30° and 33°, as compared to wild type (Figure 1A). UPRE expression three- to sevenfold higher than that of wild type was also observed at the semipermissive temperature of 30° or 33° in sec15ts-1, sec2ts-59, sec1ts-1, sec6ts-4, and sec4ts-8, which are defective in distal secretion. The pep12 strain, which is defective in vacuolar targeting and is not temperature sensitive, exhibited UPRE expression levels approximately fourfold higher than wild-type grown under identical conditions at all temperatures tested (Figure 1A). Unlike all of the other Sec– strains examined here, the sec14ts-3 single-mutant strain exhibited levels of UPRE-CYC-lacZ expression indistinguishable from wild type when shifted to temperatures of 30° and 33° (data not shown), semipermissive temperatures at which we observed substantial UPRE induction in the sec14ts-3 cki1 strain in a previous study (CHANG et al. 2002).
The mechanism of UPR activation involves splicing of the HAC1 transcript by Ire1p, following activation of Ire1p by autophosphorylation (COX et al. 1993; MORI et al. 1993; COX and WALTER 1996). To determine whether HAC1 splicing was elevated under secretory stress, Northern blot analysis was performed on RNA extracted from sec13ts-1 cells. As a control, wild-type cells were treated with 1 mM tunicamycin, which has been shown to elicit the UPR (COX and WALTER 1996). Untreated wild-type and sec13ts-1 cells grown at 25° contained only the full-length (unspliced) HAC1 transcript (Figure 1C). As expected, tunicamycin-treated wild-type cells contained two distinctive sizes of HAC1 mRNA corresponding to unspliced (HAC1u) and spliced (HAC1i) mRNA (Figure 1C). sec13ts-1 cells grown at the semipermissive temperature of 30°, in the absence of tunicamycin, likewise contained both the spliced and unspliced forms of HAC1 transcript (Figure 1C), confirming that HAC1 mRNA is actively spliced in sec13ts-1 cells at 30°.
We observed a modest increase in the expression of ß-galactosidase from the UPRE-CYC-lacZ construct after shifting wild-type cells to 36° (data not shown). Since changes in growth temperature are known to cause changes in metabolism, including alterations in the pattern of turnover of membrane lipids (DOWD et al. 2001), we examined activation of the UPR in strains treated with or without the fungal metabolite BFA at 30°. BFA blocks secretion, results in loss of COPI coats from the Golgi, and causes redistribution of Golgi enzymes to the ER (LIPPINCOTT-SCHWARTZ et al. 1989; KLAUSNER et al. 1992; KREIS et al. 1995; SCIAKY et al. 1997; SEEMANN et al. 2000). Unlike mammalian cells, most Saccharomyces cerevisiae strains are insensitive to BFA. However, erg6 mutants, which are defective in biosynthesis of ergosterol, exhibit sensitivity to BFA presumably due to increased membrane permeability (GRAHAM et al. 1993; SHAH and KLAUSNER 1993; VOGEL et al. 1993). As shown in Figure 1B, wild-type cells that are impermeable to BFA exhibited no UPRE induction in the presence of BFA at 30°. erg6 cells, however, exhibited high levels of UPRE-driven ß-galactosidase expression at 30° in medium containing 25 and 50 mM BFA (Figure 1B), concentrations that do not inhibit growth of erg6. Expression driven by the UPRE construct in the erg6 strain was elevated 
9-fold within 1 hr following exposure to 25 mM BFA (data not shown), compared to erg6 cells grown without BFA. After 2 hr of exposure to 25 mM BFA at 30°, UPRE expression was elevated 15-fold in the erg6 strain (Figure 1B). Similar elevated levels of UPRE-driven ß-galactosidase expression were observed at equivalent time points in erg6 cells grown in medium containing 50 mM BFA (Figure 1B).
INO1 expression is reduced in Sec– mutants experiencing secretory stress:
INO1 expression was assayed in a selection of the Sec– mutants as described in MATERIALS AND METHODS using an INO1-CYC-lacZ reporter gene. All of the mutants tested exhibited reduced INO1 expression compared to wild type when cultured at semipermissive growth temperatures in inositol-free (I–) medium (Figure 2A). This effect was most pronounced in the sec13ts-1 and sec16ts-2 strains, where a three- to fivefold reduction in INO1-CYC-lacZ expression was observed in cells grown in I– medium shifted to 30° compared to the same cells grown continuously at 25° (Figure 2A). The sec18ts-1, sec22ts-3, and sec21ts-1 mutants also exhibited levels of INO1 expression that were 50% of wild type or lower, even at the permissive temperature of 25° (Figure 2A). A further reduction in INO1-CYC-lacZ expression was observed in these strains when they were shifted to the semipermissive temperatures of 30° or 33°. Of the strains tested (Figure 2), the sec22ts-3 strain showed the lowest level of INO1 expression at 30° (10% of wild-type levels; Figure 2A). Somewhat reduced levels of INO1 expression were also observed at all temperatures in the sec1ts-1, sec6ts-4, and sec15ts-1 strains. In these cases, ß-galactosidase activity from the INO1-CYC-lacZ reporter gene was reduced compared to wild type even at 25°, but was not significantly further reduced at 30° or 33°. In the sec14ts-3 strain, expression from the INO1 reporter gene was reduced compared to wild type at 25° and 30°. Expression of INO1-CYC-lacZ increased somewhat after a shift to 33°, but did not reach the level of expression observed in the wild-type strain grown under comparable circumstances (Figure 2A).
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In contrast to wild type, at 30°, sec13ts-1 cells exhibited high levels of ß-galactosidase expression from the UPRE reporter gene whether inositol was present or not (Figure 2B). In fact, the level of UPRE expression in sec13ts-1 cells grown at 30° in I+ medium, exceeded the level of UPRE expression in wild-type cells shifted to I– medium by >8-fold. Following the shift to I– medium, a further increase of 30% in expression from the UPRE-CYC-lacZ reporter gene was observed in sec13ts-1 cells (Figure 2B). Overall, the level of ß-galactosidase activity driven by the UPRE construct in sec13ts-1 cells grown at 30° in I– medium exceeded that observed under the same conditions in wild-type cells by 12-fold (Figure 2B). In contrast to the high level of UPRE expression observed in sec13ts-1 cells grown at 30° in I+ medium (Figure 2B), INO1-CYC-lacZ expression in the sec13ts-1 cells at 30° was fully repressed when inositol was present (Figure 2C). When sec13ts-1 cells were shifted to I– medium at 30°, INO1-CYC-lacZ derepression occurred, but the level of ß-galactosidase activity achieved was only about one-third of the level seen in the wild-type strain (Figure 2C).
The hac1 and ire1 mutations exhibit partial synthetic lethality with mutations conferring defects in membrane trafficking:
Sec– ire1 and Sec– hac1 strains were generated for each of the Sec– mutants used in this study and, in most cases, double-mutant progeny were found to have growth defects that were more extreme than those of the corresponding parental Sec– single mutant. Examples of Sec– ire1 double mutants, from each of the categories of Sec– mutants used for the temperature-shift experiments described above, are shown in Figure 3A. Sec– hac1 double-mutant strains, which are not shown, had phenotypes very similar to the corresponding Sec– ire1 strains. No change was detected in the growth pattern on YEPD plates in response to temperature of double mutants involving sec1ts-1 (Figure 3A) or pep12 with ire1 or hac1, and slight, if any, difference was observed in the case of the double mutants involving sec4ts-8, sec14ts-3, and sec15ts-1, as compared to the corresponding single mutants (data not shown). Double mutants involving sec2ts-59 and sec21ts-1 were not generated. In all other cases, the double mutants, Sec– ire1 or Sec– hac1, failed to grow at a temperature 2°–3° lower than that of the corresponding Sec– parent (Figure 3A; Sec– hac1 data not shown).
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and hac1. For example, the sec13ts-1 ire1 and sec13ts-1 hac1 strains failed to grow at a temperature of 30° or higher, while the restrictive temperature for the sec13ts-1 strain is 33° (Figure 3A). The sec16ts-2 ire1 and sec16ts-2 hac1 strains grew poorly even at 25°, while the sec16ts-2 parental strain was able to grow, although poorly, at temperatures up to 30° (Figure 3A). Double mutants simultaneously defective in the UPR and sec12ts-4 (Figure 3A) or sec23ts-1 (data not shown) also failed to grow at temperatures 2° lower than the restrictive temperature for the corresponding parental Sec– strain. The sec23ts-1 ire1 and sec23ts-1 hac1 double mutants exhibited particularly poor growth and could grow only at 20° or lower on YEPD plates. They could not be cultured in liquid YEPD medium even at 20° and they failed to grow entirely on YEPD plates at 25° or higher. The sec23ts-1 parental strain, by contrast, was capable of normal growth on YEPD plates at temperatures up to 28° (data not shown). Double mutants carrying ire1 or hac1 in combination with mutations affecting vesicle docking and fusion, sec17ts-1 (Figure 3A), sec18ts-1, or sec22ts-3 (data not shown), also failed to grow at temperatures 2°–3° lower than the minimum restrictive temperature of the corresponding parental Sec– mutant. Among the mutations that affect distal secretion from the Golgi to the plasma membrane, only sec6ts-4 exhibited a clear negative interaction with ire1 or hac1. The sec6ts-4 ire1 and sec6ts-4 hac1 strains failed to grow at 33°, whereas the sec6ts-4 parental strain has a restrictive temperature of 35° (Figure 3A).
The ire1 and hac1 mutations were also crossed into the erg6 genetic background to assess their effect on BFA sensitivity. While growth of the erg6 parental strain was completely inhibited by the presence of 100 mM BFA, as previously reported (GRAHAM et al. 1993; SHAH and KLAUSNER 1993; VOGEL et al. 1993), and was unaffected by 25 or 35 mM BFA in YEPD medium (Figure 4), the erg6 ire1 and erg6 hac1 double-mutant strains were inhibited by 35 mM BFA (Figure 4). Thus, the presence of a UPR mutation results in an increased sensitivity of erg6 to BFA.
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(Figure 3B) and hac1 strains (data not shown) exhibit somewhat "leaky" inositol auxotrophy (Ino– phenotype; Figure 3B). The sec13-1 strain also has a leaky Ino– phenotype at its semipermissive temperature of 30° (Figure 3B) as reported by GILSTRING et al. (1999). The sec14ts-3 strain also grows more poorly in I– medium at the semipermissive temperature of 33° (Figure 3B), as reported by KEARNS et al. (1997) and CHANG et al. (2002). In a number of cases tested, the double mutants (i.e., Sec– ire1 or Sec– hac1) exhibited somewhat tighter Ino– phenotypes than either single-mutant parent did (Figure 3B). Growth of five such double mutants, sec14 ts-3, sec13ts-1, sec6ts-4, sec16ts-2, and sec17ts-1, in combination with ire1 is shown in Figure 3B.
Constitutive activation of the UPR by expression of the HAC1i gene rescues growth defects of some Sec– mutants:
The sec1ts-1, sec4ts-8, sec13ts-1, sec13ts-4, sec14ts-3, sec15ts-1, and sec22ts-3 strains were transformed with the HAC1i construct, lacking the intron sequence. The lack of the intron in HAC1i results in constitutive activation of the UPR, even in ire1 strains (COX and WALTER 1996; KAWAHARA et al. 1997, 1998; MORI et al. 2000).
The sec15ts-1 and sec14ts-3 mutants transformed with pHAC1i appeared to grow more poorly than the same strains carrying vector alone (data not shown), a result consistent with the reports of KAWAHARA et al. (1997) and MORI et al. (2000), who observed that wild-type strains transformed with pHAC1i exhibited slower growth than controls. No change was observed in the growth of the sec1ts-1, sec22ts-3, or sec4ts-8 strains when transformed with pHAC1i (data not shown). While the growth of the sec1ts-1 strain was not affected by transformation with pHAC1i (data not shown), the sec1ts-1 ire1 strain transformed with pHAC1i was able to grow at 33° (Figure 5B), a temperature at which the sec1ts-1 single mutant is unable to grow (see Figure 3A). This result was not expected, since the sec1ts-1 ire1 and sec1ts-1 strains had similar phenotypes at 33° (Figure 3).
The most dramatic effects of expression of pHAC1i were obtained in the sec13ts-1 and sec13ts-4 strains. When transformed with pHAC1i, the sec13ts-4 and sec13ts-4 hac1 strains were able to grow at 37° (Figure 5A), a temperature that is restrictive for sec13ts-4. The sec13ts-1 strain was also able to grow at 32° when transformed with pHAC1i (data not shown), whereas sec13ts-1 will not grow above its semipermissive temperature of 30° when transformed with vector alone. HAC1i was also able to rescue growth of sec13ts-1 in the absence of inositol at 30° and 32° (Figure 5C).
However, HAC1i was not able to restore sec13ts-1 growth under any condition (with or without inositol) at temperatures of 33° or greater (Figure 5C), suggesting that HAC1i expression cannot suppress complete loss of Sec13p function. To test this idea, a sec13 strain carrying the wild-type SEC13 gene on plasmid pRS316 was transformed with pHAC1i or an empty vector (YCp50) and a plasmid "shuffling" experiment was performed, as described in MATERIALS AND METHODS. When the transformants were forced to lose pSEC13, they failed to grow even when pHAC1i was present (data not shown), indicating that expression of the HAC1i gene cannot compensate for the total loss of Sec13p.
A functional UPR pathway improves the kinetics of transport to the vacuole in Sec– mutants:
To assess the effect of the UPR on membrane trafficking, we monitored the kinetics of processing in several Sec– mutants, with and without a deletion in either IRE1 or HAC1. These experiments were conducted at temperatures in which the UPR is activated in the corresponding Sec– mutant, as determined in the experiments depicted in Figure 1A. In each case, the temperature selected was permissive or semipermissive for the corresponding Sec– mutant, but restrictive in strains carrying the given Sec– mutation in combination with ire1 or hac1 (i.e., the specific Sec–ire1 or Sec–hac1 strains were incapable of sustained growth at the chosen temperature). The strains tested included sec13ts-1, sec13ts-4, sec22ts-3, and sec17ts-1 in combination with ire1 or hac1. Data are shown in Figure 5 for strains carrying sec13ts-1 and sec13ts-4 mutations. Cells were briefly shifted to the elevated temperature, as described in MATERIALS AND METHODS, and the kinetics of CPY maturation were monitored in strains using pulse-chase analysis followed by CPY immunoprecipitation and SDS-PAGE analysis followed by image analysis of the autoradiograms (Figure 6).
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strain, which is not capable of sustained growth at 30°, the kinetics of CPY processing at 30° were slowed still further as compared to both the wild-type and sec13ts-1 strains at 30°. In this strain, after 10 min, less than one-quarter of labeled CPY was present in mCPY (Figure 6, A and C). Comparable slowing in the appearance of mCPY was observed in the sec17ts-1 ire1 and sec22ts-3 ire1 strains relative to the sec17ts-1 and sec22ts-3 strains, respectively (data not shown), suggesting that slowing of CPY processing is not unique to the sec13ts-1 ire1 strain. Moreover, the kinetics of processing in an ire1 strain were identical to wild type (data not shown), indicating that the UPR pathway defect does not appreciably affect normal membrane trafficking in the absence of secretory stress.
We also measured the time course of CPY processing in the sec13ts-4 hac1 strain expressing the HAC1i gene from a centromeric plasmid and compared it to processing in the same strain transformed with an empty vector (Figure 6B). Transformants were briefly shifted to 35°, conditions in which the HAC1i gene product rescues the growth defect of sec13ts-4 hac1 strains (Figure 5A). Transformants were then subjected to pulse-chase analysis as described above. In the sec13ts-4 hac1 strain transformed with an empty vector, most of the labeled CPY remained in the ER (p1) form throughout the time course (Figure 6, B and C). However, in the sec13ts-4 hac1 strain transformed with the HAC1i gene, the kinetics of CPY processing were markedly faster than in the control transformed with vector alone (Figure 6, B and C). After 10 min, about one-third of the labeled CPY was present in mCPY compared to <20% in transformants containing vector alone (Figure 6, B and C). After 30 min, less than half of labeled CPY was processed to m in the transformants carrying vector alone, whereas in sec13ts-4 hac1 cells transformed with pHAC1i, 80% of the label was present in mCPY after 30 min.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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cells challenged with BFA (Figure 1). Moreover, our results indicate that activation of the UPR plays a functional role during secretory stress, facilitating protein trafficking in cells experiencing partial impairment in secretory function. This conclusion is supported by the partial synthetic lethality of the ire1 and hac1 mutations in combination with a large majority of Sec– mutants tested in this study (Figure 3), as well as the partial suppression of certain Sec– phenotypes by HAC1i (Figure 5). The slowing of CPY processing in Sec– strains carrying a UPR mutation and the rescue of these defects by HAC1i provide direct confirmation that UPR plays a role in facilitating protein processing under these conditions (Figure 6). However, under conditions of secretory stress created by elevating Sec– mutants to a semipermissive temperature, the observed activation of the UPR does not result in elevated INO1 expression compared to wild type, even when inositol is absent. Indeed, under conditions of secretory stress, in Sec– cells exposed to semipermissive growth conditions, INO1 expression is actually reduced compared to wild type (Figure 2) and some strains, such as sec13ts-1 and sec14ts-3 grown at their semipermissive temperatures, actually require inositol for growth (Figure 3B). Thus, it appears that the activation of the UPR, which occurs under conditions of secretory stress, is not correlated with elevated INO1 expression.
The role of the UPR and the secretory pathway in expression and regulation of the INO1 gene:
INO1 is one of a large number of coregulated genes, which contain the promoter element UASINO and respond to the availability of inositol in the growth medium (CARMAN and HENRY 1999). While the mechanism by which the cell regulates INO1 in response to inositol and other precursors of lipid metabolism has not been fully elucidated, it is known that the Ino2p and Ino4p transcription factors bind to UASINO to activate transcription (CARMAN and HENRY 1989; LOPES et al. 1991; AMBROZIAK and HENRY 1994; SCHWANK et al. 1995; GREENBERG and LOPES 1996). Furthermore, the negative regulator, Opi1p, is required for repression of UASINO-containing genes in response to inositol (WHITE et al. 1991). Recent evidence suggests that changes in the pattern of membrane phospholipid synthesis, produced in response to the incorporation of exogenous inositol, result in translocation of the negative regulator, Opi1p, to the nucleus from the endoplasmic reticulum and that Opi1p translocation coincides with repression of INO1 (LOEWEN et al. 2004).
Cox et al. (1997) suggested that activation of INO1 transcription in the absence of inositol might be linked to activation of the UPR signal transduction pathway. In support of this hypothesis, Cox et al. (1997) showed that the UPR is activated in wild-type cells growing in inositol-free medium and that deletion of OPI1 suppressed the Ino– phenotype of ire1 and hac1 mutants. Previously, consistent with the hypothesis of Cox et al., we reported that expression of both UPR and INO1 is elevated in sec14-3ts strains carrying the cki1 suppressor (CHANG et al. 2002). However, deletion of HAC1 or IRE1 did not eliminate overexpression of INO1 in sec14ts cki1 cells (CHANG et al. 2002), suggesting that activation of the UPR, while correlated with INO1 expression, is not obligatory for INO1 activation.
It has been assumed that the Ino– phenotype of UPR mutants, such as hac1 and ire1, is due to the inability to maintain wild-type INO1 expression levels in the absence of UPR activation (COX et al. 1997; CHANG et al. 2002). Another possibility is that inositol deprivation results in a stress condition that elicits UPR activation and that UPR activation under these circumstances is essential in much the same way that it is in wild-type cells experiencing stress due to the buildup of unfolded proteins following exposure to tunicamycin. We propose that providing inositol to UPR mutants under such circumstances alleviates the underlying stress condition that necessitates UPR activation for survival, thus explaining the Ino– phenotype. Since inositol limitation (COX et al. 1997; CHANG et al. 2002) and secretory stress (Figure 1) both result in UPR activation, we questioned whether these two stress conditions might have additive effects upon the level of UPRE and/or INO1 expression. Indeed, shifting the sec13ts-1 strain to inositol-free medium at the semipermissive temperature of 30° resulted in an increase of 30% in UPRE expression over the already elevated levels seen in this strain at 30° in the presence of inositol (Figure 2).
Inositol limitation and secretory stress, thus, affect UPR activation in an additive or synergistic fashion. In Sec– cells growing near their restrictive temperatures, the additive stress caused by lack of inositol may, in some cases, exceed the stress tolerance of even those cells with an intact UPR. Provision of inositol under such circumstances would reduce the stress that results in UPR induction, potentially explaining the conditional Ino– phenotype of sec13ts-1 cells near their restrictive temperature. Consistent with this idea, transformation of sec13ts-1 with HAC1i permits growth at temperatures up to 32° with or without inositol (Figure 5), whereas the parent sec13ts-1 strain transformed with the vector grows well only up to 30° and only if inositol is present (Figure 5).
While inositol limitation and secretory stress had an additive effect upon UPRE expression levels, no correlated additive effect on INO1-CYC-lacZ expression was observed in sec13ts-1 cells grown in the absence of inositol at semipermissive temperatures (Figure 2A). To the contrary, in sec13ts-1 cells shifted to 30° in the absence of inositol, INO1-CYC-lacZ expression was reduced to less than one-half of the level observed in the wild-type control grown under identical conditions (Figure 2C). Similar reductions in INO1-CYC-lacZ expression levels, relative to the wild-type control were observed in all of the Sec– mutants assayed after growth in inositol-free media at semipermissive conditions (Figure 2A). The absence of a correlation between UPRE and INO1 expression was even more apparent in sec13ts-1 cells grown at 30° in the presence of inositol (compare Figure 2B and 2C). Under these growth conditions, UPRE expression was greatly elevated in sec13ts-1 cells (Figure 2B). Yet, INO1-lacZ expression in the sec13ts-1 strain at 30° was fully repressed when inositol was present (Figure 2C). Clearly, activation of the UPR during secretory stress does not result in activation of INO1 when inositol is present.
The sec14-3ts mutation, unlike the other Sec– mutations analyzed here, does not affect an immediate component of the secretory apparatus itself. Rather, Sec14p is a lipid transfer protein that binds both phosphatidylinositol and phosphatidylcholine (AITKEN et al. 1990). sec14 mutations result in a wide range of changes in both lipid metabolism and membrane trafficking (NOVICK et al. 1980, 1981; BANKAITIS et al. 1989; AITKEN et al. 1990; CLEVES et al. 1991; KEARNS et al. 1997; PATTON-VOGT et al. 1997; HENRY and PATTON-VOGT 1998; SREENIVAS et al. 1998; XIE et al. 1998). Surprisingly, despite the wide range of Sec– mutants that exhibited UPRE activation at their semipermissive temperatures, we did not observe this effect in the sec14-3ts single mutant. The failure to observe UPRE activation in the sec14-3ts strain at the semipermissive temperatures of 30° and 33° was very surprising given that high levels of UPRE activation were previously observed at both 30° and 37° (CHANG et al. 2002) in sec14-3ts strains carrying the bypass suppressor, cki1. INADA and GUTHRIE (2004), however, recently reported active splicing of HAC1 mRNA, indicative of UPR activation, in the sec14-3ts mutant shifted to its restrictive temperature. Thus, it is possible that we failed to observe UPR activation in the sec14-3ts mutant because we used a less direct assay (i.e., a UPRE reporter construct vs. HAC1 mRNA splicing) and different growth conditions (i.e., semipermissive temperature vs. a transient shift to the restrictive condition) than did INADA and GUTHRIE (2004). At 30° and 37°, the sec14-3ts cki1 double mutant exhibits high levels of INO1 expression and inositol prototrophy and, in fact, overproduces and excretes inositol into the growth medium (PATTON-VOGT et al. 1997; CHANG et al. 2002). In contrast, the sec14-3ts single mutant exhibits inositol auxotrophy and lowered INO1 expression at the semipermissive temperature of 33°. The reduction in INO1 expression in sec14ts-3 under semipermissive growth conditions was similar to that in other Sec– strains studied here, suggesting that lowered INO1 expression is a general response to impairment of secretory function. The elevated INO1 expression that we reported previously in the sec14ts-3 cki1 strain, and other sec14 strains carrying bypass suppressors affecting the CDP-choline pathway for phosphatidylcholine synthesis, is presumably a consequence of the specific mechanism of suppression (PATTON-VOGT et al. 1997; HENRY and PATTON-VOGT 1998; CHANG et al. 2002).
Regardless of the explanation for the general lack of correlation between UPR activation and INO1 expression in Sec– mutants, it is clear that INO1 expression is reduced in Sec– mutants experiencing secretory stress. The inhibitory effect of secretory stress on transcription of INO1 is similar to the effect of secretory stress on transcription of rRNA and ribosomal protein genes. MIZUTA and WARNER (1994) showed that the function of the entire secretory pathway is essential for ribosomal synthesis. NIERRAS and WARNER (1999) subsequently demonstrated that rRNA transcription and ribosomal protein synthesis are slowed in cells undergoing secretory stress and that this response is not transduced by the UPR. Rather, they concluded that the effect on rRNA and ribosomal proteins genes in cells with secretory defects may be controlled by a mechanism involving the protein kinase C signal transduction pathway related to membrane stress. In similar fashion, under conditions of secretory stress, signals other than those generated by the UPR may take precedence in controlling expression of INO1 and other UASINO-containing genes. INO1 expression is known to be influenced by growth phase and nutrient availability GRIAC and HENRY (1999) and the glucose response pathway has also been shown to influence the levels of INO1 expression (OUYANG et al. 1999; SHIRRA and ARNDT 1999; SHIRRA et al. 2001). For these reasons, we are currently examining the relative roles of several other signal transduction pathways in transducing signals from the secretory pathway to the regulatory apparatus controlling INO1 transcription.
The UPR plays a functional role in cells experiencing secretory stress:
The idea that the UPR might play a functional role in exit from the ER and/or be induced by any slowing of ER-specific steps seems quite logical given that the UPR is known to regulate gene expression in response to stress in the ER. Several previous studies have suggested that the UPR might be activated in mutants having defects in specific early steps in membrane trafficking in yeast. For example, ROSE et al. (1989) detected an increased level of KAR2 mRNA in the sec18ts-1 mutant, which is defective in vesicle fusion and exit from the ER and SEMENZA et al. (1990) reported secretion of Kar2p (BiP) from various Sec– mutants, including sec22ts-3, sec17ts-1, sec20ts-1, and sec18ts-1, at their permissive temperatures. BELDEN and BARLOWE (2001) demonstrated that the secretion of Kar2p in sec22ts-3 cells, reported by Semenza et al., is associated with strong UPR activation. Since Sec22p may function in retrograde transport of proteins from the Golgi to the ER, BELDEN and BARLOWE (2001) speculated that accumulation of secretory proteins in the ER in sec22ts-3 cells at permissive temperatures could lead to proliferation of the ER, thereby activating the UPR.
The experiments we report here using BFA-treated erg6 cells and a wide range of secretory mutants suggest that the UPR can be triggered by secretory stress induced not only by mutations affecting exit from the ER, but also by mutations affecting a number of compartments of the secretory pathway. In the case of mutants such as sec12ts-4, sec13ts-1, sec13ts-4, sec16ts-2, and sec23ts-1, which have defects in COPII vesicle formation, and in erg6 cells treated with BFA, UPR activation might be triggered by disruption of the ER organization and accumulation of secretory cargo proteins as suggested by Belden and Barlowe. However, activation of the UPR in mutants defective in vacuolar targeting (pep12) and the Sec6p complex (Figure 1A) indicates that secretory stress induced in compartments distal to the ER is also able to elicit the UPR through Ire1p, a kinase believed to be localized exclusively in the ER (COX et al. 1993). In a related recent report, LEBER et al. (2004) have shown that ER-distal stress boosts HAC1 mRNA abundance.
TRAVERS et al. (2000) demonstrated that activation of the UPR affects expression of genes controlling a broad array of ER and secretory functions including ER-associated protein degradation (ERAD), ER-to-Golgi transport, Golgi-to-ER retrieval, vacuolar targeting, distal secretion, and cell wall biogenesis. Travers et al. also showed that the UPR was activated in ERAD mutants and that ire1 and hac1 mutants exhibit synthetic lethality with ERAD mutations. Moreover, CALDWELL et al. (2001) and VASHIST et al. (2001) demonstrated that degradation of soluble substrates by ERAD requires ER-Golgi transport, suggesting a functional relationship involving ERAD and membrane trafficking from the ER.
We have demonstrated similar synthetic lethality involving UPR and secretory mutations. In most cases, Sec– Upr– double mutants had more severe growth phenotypes than the corresponding Sec– parent. These results are consistent with the hypothesis that UPR induction provides protection to cells experiencing stress resulting from partial impairment of membrane trafficking and/or inositol limitation. Also consistent with this hypothesis is the partial suppression (i.e., elevation of the restrictive temperature by several degrees and alleviation of the Ino– phenotype) of sec13ts-1 by transformation with HAC1i (Figure 5). Consistent with the results reported here, HIGASHIO and KOHNO (2002) and SATO et al. (2002) recently reported partial suppression by transformation with pHAC1i of growth defects of the sec24ts-20 and sec12ts-4 mutants, respectively, which are defective in COPII vesicle formation. Furthermore, processing of was enhanced at the semipermissive temperature in Sec– cells retaining an active UPR pathway or carrying HAC1i, as compared to Sec– cells carrying a UPR mutation (Figure 6). Thus, the UPR not only is activated under secretory stress, but also plays a role in facilitating membrane trafficking and cell survival under these conditions.
By what mechanism might secretory stress signals generated in membrane compartments, other than the ER, result in UPR activation? One possibility is that disruption of post-Golgi trafficking perturbs the balance of anterograde and retrograde membrane transport pathways between the ER and Golgi by affecting the rate and/or efficiency of protein sorting at the Golgi. It has been suggested that for protein sorting in late-Golgi compartments, anterograde and retrograde transport pathways coexist and compete with each other (COLE et al. 1998). In mammalian cells, it has been shown that Golgi resident proteins continually cycle through the ER (COLE et al. 1998; MILES et al. 2001; WARD et al. 2001). It has been suggested that one function for this recycling is to allow Golgi residents to be periodically surveyed by the protein-folding machinery in the ER where they could be either refolded or degraded through the ERAD pathway (COLE et al. 1998; NG et al. 2000). When anterograde pathways are blocked or slowed, retrograde pathways predominate, leading to a return of secretory and resident proteins to the E
