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Address correspondence to David I. Meyer, Department of Biological Chemistry, University of California at Los Angeles School of Medicine, Los Angeles, CA 90024-1737. Tel.: (310) 206-3122. Fax: (310) 206-5197. E-mail: dimeyer{at}ucla.edu
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Abstract |
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Key Words: secretion; endoplasmic reticulum; yeast; mRNA; membrane biogenesis
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Introduction |
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The proliferation of certain ER-specific proteins results from disturbances within the lumen of the ER sensed via a well-characterized mechanism known as the unfolded protein response (UPR).* The UPR represents a signal transduction pathway in yeast that links stress in the ER to transcription of genes encoding ER-resident proteins (Patil and Walter, 2001; Spear and Ng, 2001). Several similarities exist between induction of the UPR and proliferation of ER membranes upon expression of p180. In both processes, the levels of mRNAs that encode ER-localized chaperones and phospholipid biosynthetic genes are elevated (Cox et al., 1997; Becker et al., 1999; Block-Alper et al., 2002). Furthermore, p180-induced membrane proliferation is accompanied by increased levels of secretory pathway mRNAs, another feature of cells undergoing a UPR (Becker et al., 1999; Travers et al., 2000). Increased secretory capacity, a prominent feature of p180 expression, has not been reported as a consequence of the UPR.
Cells that are deleted for IRE1 are unable to undergo a UPR. Through analysis of such mutants, the UPR has been linked to membrane-related cellular processes such as phospholipid synthesis, protein modification and secretion, and ER-associated protein degradation (Cox et al., 1997; Friedlander et al., 2000; Travers et al., 2000). These mutational analyses, coupled with the observation that levels of ER-localized proteins were elevated in the UPR, led us to investigate the relevance of the UPR to membrane proliferation stimulated by p180 expression in yeast. We conclusively demonstrate in this study that p180-induced membrane proliferation, as well as increasing mRNA levels of ER-localized proteins and phospholipid biosynthetic enzymes, occurs by an Ire1p-independent mechanism.
Instead, we report here the involvement of a novel mechanism that potentially regulates the increased abundance of secretory pathway component transcripts observed during p180-induced membrane proliferation. Our results show that p180 expression stabilizes secretory component mRNAs, and that targeting of any mRNA to the ER membrane is capable of mediating a substantial increase in its longevity. The conclusions we draw from our work in this model system could provide significant insights into processes occurring during the terminal differentiation of mammalian secretory tissues.
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Results |
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Genes whose transcription is induced by the UPR contain a 22-base pair UPR responsive element (UPRE) in their upstream regulatory regions (Mori et al., 1992; Kohno et al., 1993). To confirm that the expression of p180 does not induce the UPR, assays measuring activity at this 22-base pair UPRE were performed. The plasmid pMCZ-Y, which encodes a fusion between the UPRE of the KAR2 gene, minimal CYC1 promoter elements and the coding sequence for the Escherichia coli ß-galactosidase enzyme, has been used previously for such assays (Mori et al., 1996). As expected in cells harboring pMCZ-Y, increased ß-galactosidase activity was seen as a result of tunicamycin treatment. In extracts from cells transformed with the pYEX-BX vector and pMCZ-Y, ß-galactosidase activity increased approximately twofold after 2 h treatment with 2 µg/ml tunicamycin (Fig. 3). In contrast, expression of CT in pMCZ-Yharboring cells slightly lowered ß-galactosidase activity.
CT-expressing cells did not show a compromised UPR, as ß-galactosidase activity was increased approximately twofold in extracts from tunicamycin-treated
CT cells compared with untreated
CT cells. Together, these data provide conclusive evidence that expression of
CT does not induce the unfolded protein response, nor is the UPR required for the p180-associated increases in levels of secretory pathway mRNA or intracellular membrane proliferation.
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P180 stabilizes mRNAs that are targeted to the secretory pathway
To directly measure the turnover of the SEC61 mRNA, transcription of the endogenous SEC61 gene was placed under the control of a tetracycline-repressible promoter, creating a means to halt transcription and monitor decay of this mRNA species. The entire DNA sequence between the start codon of the upstream divergently transcribed CSR1 gene and the SEC61 start codon was replaced with elements allowing control of SEC61 expression by tetracycline. This tet-SEC61 strain produced 90% of wild-type levels of SEC61 mRNA in the absence of added drug (unpublished data). Upon addition of the tetracycline analogue, doxycyline, transcription of the SEC61 gene ceases. Decay of the SEC61 mRNA was monitored by quantitative Northern blotting in a time-course after drug addition in cells expressing the
CT construct and vector control cells. PGK1 mRNA is a housekeeping gene whose expression is not affected by p180 and was thus used as a control (LaGrandeur and Parker, 1999). In vector control cells, the half-life of SEC61 mRNA was
5 min, whereas in
CT-expressing cells, nearly all of the SEC61 mRNA remained after 40 min (Fig. 5 A). This value may be conservative, as one of our experiments indicated the half-life for SEC61 mRNA in
CT-expressing cells was in excess of 100 min (unpublished data). To verify that ribosome binding activity was integral to mRNA stabilization, we performed the same experiments in strains expressing full-length p180, which has ribosome binding activity, and in two strains that do not, namely Membrane Anchor and
NT (Fig. 1 A), as well as in
CT and vector-only strains. As can be seen in Fig. 5 B, both
CT and full-length p180-expressing strains showed a significant prolongation of SEC61 mRNA half-life (t1/2 > 45 min in both cases), whereas strains expressing constructs that do not bind ribosomes were essentially similar to controls (t1/2 for
NT, 6 min; Membrane Anchor, 7 min; and vector only, 5 min). Together, these data demonstrate a profound increase in the stability of SEC61 mRNA in p180 and
CT-expressing yeast.
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Discussion |
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In contrast to the induction of ER chaperones such as KAR2 in response to ER stress, the requirement for IRE1 for membrane proliferation is a subject of some debate. One group observed that IRE1 was essential for KAR2 mRNA induction in response to cytochrome p450 overexpression; however, it was not necessary for the production of ER-like membranes that arise upon expression of the protein (Menzel et al., 1997). A subsequent report demonstrated that IRE1 was essential for the proliferation of cytochrome p450-induced membranes, and revealed that differences in strain background accounted for the conflicting results (Takewaka et al., 1999). In our experiments, the IRE1 gene was deleted in two nonisogenic strains and found to be dispensable for p180-induced membrane proliferation and elevated levels of KAR2 (unpublished data).
Our results also address the role of Ire1p in the inositol response and in membrane biogenesis. Phosphatidylinositol is one of the major lipids of membranes in eukaryotic cells. An understanding of any type of membrane proliferation will depend on an elucidation of how the need for additional membrane lipid is met. There appear to be multiple mechanisms by which a key element in this lipid's metabolism, the INO1 gene, is regulated. It has been shown that deletion of IRE1 results in inositol auxotrophy, suggesting that the UPR and phospholipid biosynthesis may be linked (Nikawa and Yamashita, 1992). Moreover, wild-type cells that were induced to undergo a UPR had increased INO1 transcription and deletion of OPI1, a gene that encodes an inhibitor of inositol phospholipid synthesis restored inositol prototropy to the ire1 strain (Cox et al., 1997).
On the other hand, we demonstrate here that INO1 mRNA can be up-regulated in the presence of p180 in a UPR-independent fashion. Similarly, Stroobants et al. (1999) showed that the inositol response is not necessarily linked to the UPR, as ire1 cells grown in the presence of oleate as the sole carbon source were capable of undergoing membrane proliferation upon overexpression of Hmg1p and the peroxisomal membrane protein, Pex15p (Stroobants et al., 1999). Others had shown that overexpression of HMG CoA-reductase (Hmg1p), which triggers the proliferation of karmellae, impaired the growth of ire1
cells, suggesting a block in membrane biogenesis, although membrane biogenesis per se was not assessed (Cox et al., 1997). They demonstrated that the level of INO1 mRNA was activated in response to growth on oleate, dependent on the presence of oleate-specific transcriptional activators. The observation that there may be multiple mechanisms for activating INO1 is further substantiated by our observation that Ino2p, a basic helixloophelix transcription factor essential for the production of INO1 mRNA, is essential for the formation of p180-induced membranes (Block-Alper et al., 2002). How INO2 is activated in response to p180 expression is unknown; however, it likely plays a critical role in the regulation of several phospholipid biosynthetic genes involved in the proliferation of intracellular membranes. Thus, it appears that the product of the INO1 gene, inositol-1phosphate, is an important phospholipid component that is likely activated by a variety of mechanisms, Ire1p-dependent and -independent, during conditions of ER stress.
Microsomes isolated from yeast cells expressing p180 constructs that contain the ribosome binding domain (p180-FL and CT) were shown to bind 24 times as many ribosomes as control microsomes in an in vitro ribosome-binding assay (Wanker et al., 1995). The high affinity of p180 for ribosomes (Kd = 1020 nM) could account for the presence on the ER membrane of some "free polysomes" containing the normally cytosolically translated mRNAs (such as PGK1) (Fig. 7). Importantly, the overall level of PGK1 mRNA between p180-expressing and control cells remained unchanged. This suggests that mere recruitment of polysomes to the ER is not solely responsible for mRNA stabilization and that other factors are likely involved. Such factors could include targeting coupled with translation of mRNAs and/or protein translocation on the ER membrane.
Targeting of mRNA to the membrane in the presence of p180 influences its turnover. This was shown in experiments using tetracycline-repressible forms of GFP and KAR2 signal sequencemodified GFP (Fig. 6) where mRNA turnover decreased about two- to threefold. Another contributing factor would be translation on the membrane. This is supported by our data on SEC61 mRNA presented in Fig. 5, where turnover rates decreased by at least 8-fold and in other experiments by as much as 10- or 20-fold. Sec61p is a very hydrophobic poreforming protein with multiple membrane-spanning domains. A previous study using a biological assay supports at least one of our interpretations. The investigators observed that the targeting the mRNA of a cytosolically translated histone bearing a hydrophobic signal peptide prevented its otherwise rapid cell cycledependent degradation (Zambetti et al., 1990). This mislocalization of mRNAs to the ER presumably prevented their degradation by cytosolic enzymes.
In p180-expressing cells, membrane-localized mRNAs could achieve protection from degradation by their physical association with ribosomes that are tightly bound to the membrane. Another interesting possibility is that mRNAs that are targeted to the ER interact directly with p180. If such an interaction occurs, it may be with regions traditionally recognized to promote mRNA stability, such as the 3'UTR. Interestingly, Lande et al. (1975) reported that in WI-38 cells (a human fibroblast cell line with well-developed rough ER) mRNAs can remain associated with the ER membrane after the membranes have been stripped of ribosomes (Lande et al., 1975). The poly-A portion of these mRNAs remains attached even following extensive treatment with RNase. An interesting and testable hypothesis is that p180 binds elements at or near the poly-A tail of certain mRNAs, thereby increasing their association with the membrane and protecting them from degradation. Lastly, p180 could be associating with both forms of RNA, ribosomal and messenger, stabilizing the translational complex. In this way an mRNA could experience increased levels of translation, surrounded by a variety of factors that render it inaccessible to enzymes involved in its turnover.
We have recently shown that Ino2p is required for generalized membrane proliferation (Block-Alper et al., 2002). Its deletion yields strains incapable of producing membranes of any kind in response to overexpression of membrane proteins, including all forms of p180 (see Fig. 1), and Hmg1p. Yet in this study, only ribosome-binding forms of p180 were able to stabilize secretory pathway mRNAs. This suggests that at least two steps are required for the production of functional ER in yeast. The first requires an intact INO2 gene and is needed for proliferation of the lipid bilayer common to all proliferated membranes. The second mechanism, involving p180 incorporation into the bilayer, enables higher expression of key proteins that will be integrated into the organelles of the secretory pathway, rough ER being a prime example. Thus, overexpression of any one of a variety of membrane proteins will trigger membrane biogenesis, as has been observed by a number of groups. When the inducer contains a ribosome binding domain, the longevity of key mRNAs will be enhanced, leading to the production of membranes containing the appropriate machinery to participate in the secretory pathway. This is precisely what is seen in the case of the CT construct, which is little more than a membrane anchor with a ribosome binding domain. The membrane anchor confers the protein's ability to stimulate INO2-dependent bilayer formation, while the ribosome binding domain provides the most efficient stabilizer of specific mRNAs.
Thus far, these experiments do not address the role of p180 in mammalian secretory cells. It is still unknown whether the events that occur upon expression of a foreign membrane protein in yeast reproduce those that occur during terminal differentiation of cells with a high capacity for secretion. Our results, however, are promising. As the complexity and functionality of the membranes produced in response to p180 expression far exceeds that which is seen for other proteins, our studies assume a greater significance. The fact that mRNA stabilization is associated with p180 expression has generated a testable hypothesis as to how increases in secretory pathway proteins could occur in the terminal differentiation in a variety of tissues including pancreas, liver, mammary gland, and plasma cells of the immune system.
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Materials and methods |
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Growth conditions and yeast transformations
Yeast cells were grown in 4% dextrose, 0.17% Difco yeast nitrogen base without amino acids and ammonium sulfate, and 5% ammonium sulfate (all Fisher Scientific) with the addition of nucleotides and amino acids as appropriate. Transformations were performed using lithium acetate transformation procedures as described (Gietz et al., 1995). Induction of expression of p180 or its derivatives from pYEX-BX plasmids was performed by growing the cells overnight to an OD of 0.53.5, diluting the cells to an OD of 0.5, and adding of CuSO4 to 0.5 mM. Cells were induced for 57 h. Where indicated, tunicamycin (Calbiochem) was added to 2 µg/ml for 2 h and doxycycline (Sigma-Aldrich) was added to 5 µg/ml.
Plasmids
Plasmids were constructed according to standard techniques. The p180 constructs (full-length, CT,
NT, MA) in pYEX-BX have been described previously (Becker et al., 1999). pBSK-TRP1 was a gift from Greg Payne (University of California at Los Angeles, Los Angeles, CA). The SEC61-LacZ plasmid encodes DNA from -1,000 to +51 of the SEC61 gene, fused to the E. coli LacZ gene in the XhoI-XbaI sites of pRS314 (Sikorski and Hieter, 1989). pMCZ-Y was a gift from K. Mori (HSP Research Institute, Shimogyoku, Japan) (Mori et al., 1996). pCM182 was a gift from E. Herrero (Universitat de Lleida, Spain) (Gari et al., 1997). The tet-SEC61 plasmid was created by PCR amplification of a 3' truncation of the CSR1 gene followed by digestion with EcoRV (endogenous) and Bgll (primer-encoded) and a BamHI-EcoRI digest of pBKS-TRP1 followed by simultaneous ligation of these DNA fragments into the EcoRI-EcoRV sites of pCM182. Amplification of the coding sequence for SEC61 was followed by digestion with BamHI (in primer) and StuI (endogenous) and cloning of this fragment into the same sites in pCM182.
Transcription run-on assay
Transcription run-ons were performed essentially as described (Parker et al., 1991), except that ATP was added to 6 mM and phosphocreatine and creatine phosphokinase were omitted. PCR products encoding the entire open reading frames of the SEC61, PYK1, and GFP genes and from nucleotides 12002048 of the KAR2 gene were gel purified using a Gel Extraction Kit (Qiagen), precipitated, and redissolved in ddH2O. PCR products were denatured by the addition of NaOH to 125 mM and 1 µg of DNA was spotted on to Magna Membranes (Osmonics, Inc.). Radiolabeled transcripts were isolated as the supernatants from the protein precipitation step of the MasterPure genomic DNA isolation kit (Epicentre), and incubated overnight at 42 degrees with blots that had prehybridized in prehybridization/hybridization solution (Brown and MacKay, 1997). Blots were rinsed twice, washed twice for 10 min at 42 degrees and twice for 30 min at 50 degrees in 0.1x SSC, 0.5% SDS and exposed PhosphorImaging screens.
Membrane fractionation
Strains harboring the YEX vector alone or the CT construct of p180 were induced in wild-type yeast for 6 h with copper sulfate. Approximately 1,000 OD600 of each strain was harvested by centrifugation. The cells were ground with a mortar and pestle in liquid nitrogen and resuspended in 5 ml Buffer I (20 mM Hepes, pH 7.4, 100 mM potassium acetate, 2 mM magnesium acetate). The fractionation was performed as described (Stoltenburg et al., 1995), except RNA from the pellet containing the membrane-bound polysomes was released by extraction with glass beads in LETS buffer (0.1 M LiCl, 0.01 M EDTA, 0.01 M Tris/HCl, pH 7.4, 0.2% SDS) instead of 0.2% sodium deoxycholate and 0.5% Tween-20. RNA was extracted as described below.
RNA isolation and Northern blot analysis
For total cellular RNA extraction, RNA was isolated as a by-product of DNA using the MasterPure genomic DNA isolation kit (Epicentre) and dissolved in formamide. For membrane fractionation experiments, RNA was extracted several times from free and membrane-bound polysome suspensions in an equal volume of DEPC H2O-saturated phenol/choloform/iso-amylalcohol (49:49:2), precipitated overnight at -20°C in isopropanol, and dissolved in formaldehyde. Five micrograms of RNA were loaded per lane for Northern blots, except for tet-SEC61 and tet-KAR2ssGFP blots, where 3 µg RNA were used. Gel electrophoresis, capillary transfer to Magna Membranes (Osmonics) and probing and washing of the blots were performed as described (Brown and MacKay, 1997). DNA fragments used as probes were generated from yeast genomic DNA or plasmid DNA with the following oligonucleotide primers: KAR2, 5'-AACTGCAGATGTTTTTCAACAGACTAAGCGC-3' and 5'-ACGCATGTCGACCTACAATTCGTCGTGTTCGAA-3'; SEC61, 5'-CTAGCTGTCGACATGTCCTCCAACCGTGTTCTAGACT-3' and 5'-AACTGCAGTCACATCAAATCAGAAAA-TCCTGGAACG-3'; INO1, 5'-CCTTGATTTATTCTGTTTC-3' and 5'-ATCTCTCTTGGAATCTTAGTTGG-3'; PGK1, 5'-AACGTCCCATTGGACGGTAA-3' and 5'-TCTTGTCAGCAACCTTGGCA-3'; PYK1, 5'-CTAGCTGTCGAGATGTCTAGATTAGAAAGATTGACCTCATTAAAC-3' and 5'-AACTGCAGTTAAACGGTAGAGACTTGCAAAGTGTTG-3'; GFP, 5'-GGGGATCCCATGCTCGAGAGTAAAGGAGAAGAAC-3' and 5'-GTGTTTGTATAGTTCATCCATGCCATG-3'; PEP4, 5'-GCATTATTGCCATTGGCCTT-3' and 5'-GTGTCTTGAGAAATGTAACCTTCCA-3'. Radiolabeling was performed using the Random Prime DNA labeling kit (Invitrogen Life Technologies, Inc.) according to the manufacturer's instructions. Blots were exposed to PhosphorImaging screens, quantitated using ImageQuant software (Molecular Dynamics). Values reported reflect normalization to levels of PYK1 or PGK1 RNA, except for membrane fractionation experiments, where RNA levels were normalized to scanned and quantified levels methylene bluestained RNA.
ß-galactosidase assays
Quantitation of ß-galactosidase activity was performed using liquid assays with o-nitrophenyl-ß-D-galactopyranoside (ONPG) as a substrate. Yeast transformants were grown overnight in appropriate synthetic media, then diluted to 0.5 OD, and grown for 5 h with 0.5 mM CuSO4 to induce expression of p180 constructs. 10 ml cells were harvested, extracts prepared, and enzyme activity quantitated as described (Breeden and Nasmyth, 1987) with the following modifications: cells were lysed in Z-buffer without ß-mercaptoethanol and reactions were performed in a microtiter plate with 5 µl extract, 100 µl Z buffer with 1 mM DTT in place of ß-mercaptoethanol, and 0.7 mg/ml ONPG. Change in absorbance at 430 nm was measured following addition of 30 µl 1 M Na2CO3 to stop the reaction. Protein concentration was measured with the Bio-Rad Laboratories protein assay according to the manufacturer's instructions. Units are defined as 1000 x A430/(c x t x v) where c, protein concentration in mg/ml; t, time of reaction; and v, volume of extract in µl.
Electron microscopy
Yeast cells were grown to mid-log phase in the presence of copper to induce expression of p180 constructs. Approximately 108 cells were harvested, washed once in 100 mM Tris-sulfate, pH 9.0, once in 100 mM Tris-sulfate, pH 9.0 + 10 mM DTT, and once in sorbitol buffer (1.2 M sorbitol, 20 mM potassium phosphate, pH 7.4, 5 mM DTT). Cells were resuspended in spheroplasting media (SD media + 1.2 M sorbitol, 20 mM potassium phosphate, pH 7.4, 5 mM DTT) with 100 U oxalyticase and incubated at 45°C for 30 min. Cells were fixed in two steps: one 30 min incubation at 4°C in 4% glutaraldehyde solution (4% glutaraldehyde, 0.1 M cacodylic acid, 1.2 M sorbitol, 20 mM potassium phosphate, pH 7.4), and one 20 min incubation at 4°C in 2% glutaraldehyde solution (0.375 M sodium cacodylate, 3.75% sucrose, 2% glutaraldehyde). Cells were washed three times in 0.5 M sodium cacodylate, 5% sucrose, and resuspended in 2% glutaraldehyde solution. The remainder of the process was performed as described (Block-Alper et al., 2002).
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Footnotes |
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* Abbreviations used in this paper: FP, free polysomes; GFP, green fluorescent protein; MBP, membrane-bound polysomes; UPR, unfolded protein response.
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Acknowledgments |
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Submitted: 3 December 2001
Revised: 16 January 2002
Accepted: 5 February 2002
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References |
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Becker, F., L. Block-Alper, G. Nakamura, J. Harada, K.D. Wittrup, and D.I. Meyer. 1999. Expression of the 180-kD ribosome receptor induces membrane proliferation and increased secretory activity in yeast. J. Cell Biol. 146:273284.
Block-Alper, L., P. Webster, X. Zhou, L. Supekovà, W.-H. Wong, P.G. Schultz, and D.I. Meyer. 2002. IN02, a positive regulator of lipid biosynthesis, is essential for the formation of inducible membranes in yeast. Mol. Biol. Cell. 13:4051.
Breeden, L., and K. Nasmyth. 1987. Cell cycle control of the yeast HO gene: cis- and trans-acting regulators. Cell. 48:389397.[Medline]
Brown, T., and K. MacKay. 1997. Analysis of RNA by Northern and slot blot hybridization. Current Protocols in Molecular Biology. F. Ausubel, editor. John Wiley and Sons, Inc., New York. 4.9.14.9.16.
Cox, J.S., R.E. Chapman, and P. Walter. 1997. The unfolded protein response coordinates the production of endoplasmic reticulum protein and endoplasmic reticulum membrane. Mol. Biol. Cell. 8:18051814.[Abstract]
Elgersma, Y., L. Kwast, M. van den Berg, W.B. Snyder, B. Distel, S. Subramani, and H.F. Tabak. 1997. Overexpression of Pex15p, a phosphorylated peroxisomal integral membrane protein required for peroxisome assembly in S. cerevisiae, causes proliferation of the endoplasmic reticulum membrane. EMBO J. 16:73267341.
Gari, E., L. Piedrafita, M. Aldea, and E. Herrero. 1997. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast. 13:837848.[CrossRef][Medline]
Kohno, K., K. Normington, J. Sambrook, M.J. Gething, and K. Mori. 1993. The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol. Cell. Biol. 13:877890.[Abstract]
Koning, A.J., C.J. Roberts, and R.L. Wright. 1996. Different subcellular localization of Saccharomyces cerevisiae HMG-CoA reductase isozymes at elevated levels corresponds to distinct endoplasmic reticulum membrane proliferations. Mol. Biol. Cell. 7:769789.[Abstract]
LaGrandeur, T., and R. Parker. 1999. The cis acting sequences responsible for the differential decay of the unstable MFA2 and stable PGK1 transcripts in yeast include the context of the translational start codon. RNA. 5:420433.
Lande, M.A., M. Adesnik, M. Sumida, Y. Tashiro, and D.D. Sabatini. 1975. Direct association of messenger RNA with microsomal membranes in human diploid fibroblasts. J. Cell Biol. 65:513528.[Abstract]
Menzel, R., F. Vogel, E. Kargel, and W.H. Schunck. 1997. Inducible membranes in yeast: relation to the unfolded-protein-response pathway. Yeast. 13:12111229.[CrossRef][Medline]
Mori, K., A. Sant, K. Kohno, K. Normington, M.J. Gething, and J.F. Sambrook. 1992. A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J. 11:25832593.[Abstract]
Mori, K., T. Kawahara, H. Yoshida, H. Yanagi, and T. Yura. 1996. Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells. 1:803817.
Parker, R., D. Herrick, S.W. Peltz, and A. Jacobson. 1991. Measurement of mRNA decay rates in Saccharomyces cerevisiae. Methods Enzymol. 194:415423.[Medline]
Parrish, M.L., C. Sengstag, J.D. Rine, and R.L. Wright. 1995. Identification of the sequences in HMG-CoA reductase required for karmellae assembly. Mol. Biol. Cell. 6:15351547.[Abstract]
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Stoltenburg, R., T. Wartmann, I. Kunze, and G. Kunze. 1995. Reliable method to prepare RNA from free and membrane-bound polysomes from different yeast species. Biotechniques. 18:564566, 568.[Medline]
Takewaka, T., T. Zimmer, A. Hirata, A. Ohta, and M. Takagi. 1999. Null mutation in IRE1 gene inhibits overproduction of microsomal cytochrome P450Alk1 (CYP 52A3) and proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae. J. Biochem. (Tokyo). 125:507514.[Abstract]
Wanker, E.E., Y. Sun, A.J. Savitz, and D.I. Meyer. 1995. Functional characterization of the 180-kD ribosome receptor in vivo. J. Cell Biol. 130:2939.[Abstract]
Wilsbach, K., and G.S. Payne. 1993. Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO J. 12:30493059.[Abstract]
Wright, R., M. Basson, L. D'Ari, and J. Rine. 1988. Increased amounts of HMG-CoA reductase induce "karmellae": a proliferation of stacked membrane pairs surrounding the yeast nucleus. J. Cell Biol. 107:101114.[Abstract]