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Article |
Address correspondence to David J. Eide, Dept. of Nutritional Sciences, 217 Gwynn Hall, University of Missouri, Columbia, MO 65211. Tel.: (573) 882-9686. Fax: (573) 882-0185. email: eided{at}missouri.edu
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Abstract |
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Key Words: zinc; transport; cation diffusion facilitator; endoplasmic reticulum; unfolded protein response
S. Clark's present address is F032A, 800 Hospital Dr., Dept. of Medicine-Endocrinology University of Missouri-Columbia, Columbia, MO 65211.
T. Lyons' present address is 404 Leigh Hall, Dept. of Chemistry, University of Florida, 127 Chemistry Research Building, P.O. Box 117200, Gainesville, FL 32611-7200.
Abbreviations used in this paper: CDF, cation diffusion facilitator; ERAD, ER-associated degradation; GPI, glycosylphosphatidylinositol; TPEN, N,N,N',N'-tetrakis-(2-pyridyl-methyl)ethylenediamine; UPR, unfolded protein response; ZRE, zinc-responsive element.
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Introduction |
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Members of the cation diffusion facilitator (CDF) family of transport proteins are likely candidates to transport Zn2+ into the secretory pathway. CDF proteins play conserved roles in transporting Zn2+ from the cytosol into organelles or out of the cell in many organisms (Nies and Silver, 1995; Paulsen and Saier, 1997; Gaither and Eide, 2001; Palmiter and Huang, 2004). In E. coli, for example, the ZitB CDF protein transports excess zinc from the cytosol to the extracellular environment (Grass et al., 2001). In mammals, ZnT-1 similarly transports excess cytosolic zinc, whereas ZnT-2 may detoxify the metal by sequestering it in the late endosome (Palmiter and Findley, 1995; Palmiter et al., 1996; Kobayashi et al., 1999). Three mammalian CDF proteins implicated in Zn2+ transport into the secretory pathway are ZnT-5, ZnT-6, and ZnT-7, each of which having been localized to the Golgi apparatus (Huang et al., 2002; Kambe et al., 2002; Kirschke and Huang, 2003). The functional roles of these proteins are not yet clear.
We have learned much about zinc homeostasis from studies of the yeast S. cerevisiae. In this yeast, zinc uptake is mediated by the high affinity Zrt1 transporter and the lower affinity Zrt2 and Fet4 proteins (Zhao and Eide, 1996a, 1996b; Waters and Eide, 2002). The genes encoding these transporters are controlled by the Zap1 transcriptional activator protein (Zhao and Eide, 1997). Zap1 is active in zinc-limited cells and its activity is repressed in zinc-replete cells. Zap1 binds to one or more zinc-responsive elements (ZREs) in the promoters of its target genes (Zhao et al., 1998).
Zinc storage and detoxification in S. cerevisiae is mediated by the vacuole. The Zrc1 and Cot1 proteins, both members of the CDF family, are responsible for zinc transport into the vacuole (Kamizono et al., 1989; MacDiarmid et al., 2000, 2002; Miyabe et al., 2001). Two other CDF proteins, Mmt1 and Mmt2, have been implicated in iron transport in the mitochondria (Li and Kaplan, 1997). The fifth yeast CDF member, and the subject of this report, is Msc2. Msc2 was first identified in a screen for mutations with altered frequencies of meiotic sister chromatid exchange (Thompson and Stahl, 1999). However, the link between DNA recombination and the MSC2 gene is unclear; the effects on recombination are allele specific and not observed in a full msc2 deletion mutant (Thompson and Stahl, 1999). A more recent study of Msc2 suggested that it played some role in zinc metabolism (Li and Kaplan, 2000). An msc2 mutant grew poorly on respired carbon sources at elevated temperatures and had an abnormally large cell size. Both of these phenotypes were suppressed by addition of excess zinc. In this report, we demonstrate that Msc2 is an ER membrane protein whose role is to maintain proper function of the ER. Our results indicate that key processes in the ER require Zn2+ and that Msc2 is involved in supplying Zn2+ to this compartment.
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Results |
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Msc2 localizes to the ER
Induction of the UPR in low zinc suggested that zinc transport into the lumen of the ER is required for ER function. A transporter protein possibly involved in delivering zinc into the ER is encoded by the MSC2 gene. Previous studies suggested that Msc2 was localized to the endoplasmic reticulum. However, these experiments were done under conditions where Msc2 was overexpressed. Because overexpression can result in protein mislocalization, the true intracellular location of Msc2 was unclear. Therefore, we determined the localization of Msc2 when expressed from its own promoter on a low copy plasmid. To aid detection of Msc2, we generated an MSC2 allele with three HA tags fused to its COOH terminus. Immunoblots detected only a single band near the predicted molecular mass of Msc2 (unpublished data). Moreover, the epitope-tagged protein complemented the temperature-sensitive growth defect phenotype of an msc2 mutant strain indicating that it is functional.
Attempts to determine the subcellular localization of Msc2 using immunofluorescence microscopy were inconclusive because the level of expression was too low. Therefore, we used sucrose gradient fractionation to assess the distribution of Msc2. Protein extracts of wild-type cells expressing HA-tagged Msc2 were separated on sucrose gradients. After centrifugation, fractions were collected and analyzed by immunoblotting. Previous reports have shown that the presence or absence of Mg2+ greatly alters the position of ER vesicles in the gradient (Roberg et al., 1997). Without added Mg2+, the ER colocalizes with the Golgi apparatus in the middle fractions of the gradient. However, in the presence of Mg2+, the ER localizes to the heavier fractions of the gradient, colocalizing with the plasma membrane. This shift to heavier fractions is likely due to ribosomes remaining associated with the ER when Mg2+ is present. If Msc2 localizes to the ER, we predicted that the protein would show this Mg2+-dependent shift in these gradients.
Kex2 is a Golgi marker protein and its localization, peaking around fraction 6 in the middle of the gradients (Fig. 2), was unaffected by Mg2+. A plasma membrane protein, Pma1, also was largely unaffected by Mg2+ levels with its peak localization in the heaviest fractions of both gradients. Dpm1 served as an ER marker. Both Msc2 and Dpm1 showed the diagnostic ER Mg2+ shift being found in the heavier fractions with Mg2+ and localizing in the middle fractions without Mg2+. Because Msc2 colocalizes with an ER marker protein and shows the characteristic ER Mg2+ shift, these studies strongly support the localization of Msc2 to that compartment. In the presence of Mg2+, some Msc2 was also found in lighter fractions that may correspond to the Golgi. Little Dpm1 was found in these lighter fractions.
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Genetic and biochemical evidence for ER dysfunction in msc2 mutants
The requirement for an intact Ire1Hac1 signaling pathway to induce the UPR in response to low zinc and loss of Msc2 function suggested that these factors perturbed ER function. Further evidence supporting this conclusion came from the observation that msc2 ire1 and msc2 hac1 mutants exhibit a synthetic lethal growth phenotype. msc2 single mutants grow poorly at 37°C on YP medium supplemented with glycerol and ethanol, two nonfermentable carbon sources, but show no such defect on media supplemented with glucose (YPD), a fermentable carbon source (Fig. 6). Similarly, neither ire1 nor hac1 mutants exhibit a growth defect on YPD at either 30 or 37°C. Combining these mutations, i.e., msc2 ire1 and msc2 hac1, resulted in a strong growth defect at 37°C. (The few colonies seen in the msc2 hac1 mutant at 37°C may be the result of spontaneously arising suppressor mutations. More than 99% of the inoculated msc2 hac1 cells failed to grow at the elevated temperature.) Introducing the MSC2 or HAC1 genes on low copy plasmids (pMSC2, pHAC1) back into the msc2 hac1 mutant restored growth at 37°C. Thus, an msc2 mutant requires both Ire1 and Hac1 to survive at higher temperatures.
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The UPR system induces expression of Scj1 and a second, partially redundant chaperone called Jem1 (Fig. 1; Nishikawa and Endo, 1997). Although Scj1 is probably zinc dependent, Jem1 lacks the zinc-binding sites and is therefore likely to be zinc independent. Therefore, increased expression of the Jem1 chaperone may compensate for the loss of zinc-dependent Scj1 activity. To test this hypothesis, we examined CPY* degradation in jem1 mutant cells lacking the zinc-independent chaperone. CPY* turnover in a jem1 single mutant was rapid in both zinc-limited and replete cells (Fig. 7). CPY* degradation in zinc-replete jem1 msc2 mutants was indistinguishable from the jem1 single mutant. However, the jem1 msc2 mutant showed a marked defect in CPY* degradation in low zinc. These studies indicate that zinc deficiency impairs function of components, likely Scj1, of the ERAD system. Consistent with this hypothesis, CPY* degradation was defective in a jem1 scj1 double mutant at both zinc levels (Fig. 7).
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Discussion |
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Several lines of evidence support the hypothesis that Msc2 is involved in zinc metabolism. First, Msc2 is a member of the CDF family of metal ion transporters. Studies of CDF proteins in many organisms have established that these proteins transport their metal ion substrate, usually Zn2+, from the cytosol to either outside of the cell or into organelles (Nies and Silver, 1995; Paulsen and Saier, 1997; Gaither and Eide, 2001; Palmiter and Huang, 2004). Thus, Msc2 is likely to be a zinc transporter whose topology is consistent with metal transport into the ER lumen. Second, mutations in msc2 alter zinc homeostasis. Although it was shown previously that the Zap1 transcription factor was completely inactive in a zinc-limited msc2 mutant (Li and Kaplan, 2000), our experiments further clarified these earlier results by showing that Zap1 can still respond to zinc deficiency in an msc2 strain. Nonetheless, the suppression of ZRE-lacZ expression caused by the msc2 mutation that we observed (Fig. 3) is consistent with msc2 mutants having elevated pools of labile cytosolic zinc. Third, mutations in MSC2 result in several phenotypes that are zinc suppressible. Li and Kaplan (2000) had shown that the growth defect of msc2 mutants on respired carbon sources at elevated temperatures is suppressed by zinc. Here, we document that zinc-deficient cells up-regulate their UPR system and this induction is further increased in an msc2 mutant. Like the temperature-sensitive growth phenotype, induction of the UPR is zinc suppressible. Finally, we present here genetic evidence that known zinc transporters, Zrc1 and Cot1, play redundant roles with Msc2. Their involvement in ER function indicates that Msc2 is also a zinc transporter.
Our results indicate that Msc2 delivers zinc to the lumen of the ER to maintain function of that compartment and, perhaps, other organelles of the secretory pathway. Although currently available methods do not allow us to assay lumenal ER zinc directly, several observations indirectly support this hypothesis. First, we found that Msc2 localizes to the ER when expressed at physiological levels from its own promoter. The presence of significant amounts of Msc2 protein in vesicles of lighter density (Fig. 2 A, +Mg2+) suggests that some protein may also be found in later compartments of the secretory pathway, perhaps the Golgi. Second, mutation of MSC2 activates the UPR system. Our results indicate that this induction requires the full UPR signaling pathway consistent with actual ER stress being responsible rather than downstream perturbations of, for example, Hac1i activity. Third, we found that msc2 mutants show synthetic growth defects when combined with mutations in either ire1 or hac1. This observation is especially intriguing in light of studies by Ng et al. (2000) in which mutations were identified that were synthetically lethal when combined with an ire1 mutation. This screen identified 16 different genes almost all of which were shown to be involved in ER functions such as glycosylation, GPI anchor synthesis, or ERAD function. Thus, mutations in genes affecting lumenal ER processing events are lethal when combined with ire1 mutations. Although msc2 mutants were not identified in this synthetic lethal screen, they too are synthetically lethal with an ire1 mutation when cells are grown at 37°C. Finally, we directly observed zinc-suppressible defects in one aspect of ER function, ERAD. We chose to examine this process because Scj1, a chaperone protein of the ER lumen that is required for ERAD, is likely to be zinc dependent. One caveat to this experiment is that the E3 ubiquitin-protein ligases required for proteasome degradation of unfolded proteins after their export from the ER, Hrd1/Der3, and Doa10/Ssm4, may also be zinc dependent (Bordallo and Wolf 1999; Swanson et al., 2001). Therefore, the defects in ERAD observed in msc2 mutants could be due to disruption of ligase function via alterations in cytosolic zinc homeostasis. This appears not to be the case; degradation of a model cytosolic substrate of Doa10, the Deg1-ß-galactosidase protein (Swanson et al., 2001), was unaffected by either zinc limitation or mutation of msc2 (unpublished data). Compromised function of Hrd1/Der3 is still formally possible but less likely given the lack of effects on Doa10 function.
In addition to ERAD, it is also likely that other processes occurring in the ER are impaired by zinc deficiency. Several observations suggest that synthesis of GPI anchors may be disrupted by these perturbations. First, GPI anchor synthesis has been found to be zinc dependent both in vitro and in vivo (Mann and Sevlever, 2001; Sevlever et al., 2001). Second, the yeast MCD4, LAS21, and GPI13 genes encode related proteins required for GPI anchor synthesis (Gaynor et al., 1999; Tohe and Oguchi, 1999; Flury et al., 2000; Taron et al., 2000). These proteins all contain conserved domains similar to the zinc-binding sites of alkaline phosphatases (Galperin and Jedrzejas, 2001). Although the precise role of these proteins is still unclear, their importance in GPI anchor synthesis is well documented. MCD4 and GPI13 are essential genes whereas LAS21 is not. Temperature-sensitive mcd4 alleles and las21 mutants have a large cell morphology similar to that seen with msc2 (Mondesert et al., 1997; Li and Kaplan, 2000; Ni and Snyder, 2001). Suppressors of these mutations also link their activity to Msc2. The large cell phenotype of las21 is suppressed by overexpression of the HSP150 gene (Tohe and Oguchi, 1999). Hsp150 is a cell wall protein of unknown function. We have found HSP150 overexpression also suppresses the msc2 temperature-sensitive growth defect and large cell phenotype (unpublished data). Finally, MCD4 is a direct Zap1 target gene and is induced greater than or equal to fourfold by zinc deficiency (Lyons et al., 2000). These results suggest that GPI anchor synthesis is sensitive to zinc deficiency, and MCD4 is up-regulated to maintain sufficient activity in zinc-limited cells. Golgi function was unimpaired by mutation of the MSC2 gene; analysis of the kinetics of wild-type CPY processing indicated no differences between wild-type and mutant cells in high or low zinc (unpublished data).
In this work, we have identified three different transporters that likely contribute to ER zinc: Zrc1, Cot1, and Msc2. Of these three, Msc2 appears to play the predominant role because mutation of this gene alone had the strongest effect on UPR induction. Although Msc2 is resident in the ER membrane, Zrc1 and Cot1 are most abundant in the vacuolar membrane (Li and Kaplan, 1998; MacDiarmid et al., 2002). We can suggest two possible mechanisms to explain how Zrc1 and Cot1 could supply zinc to the ER. First, these proteins may mediate zinc transport soon after insertion into the ER membrane and before their transit to the vacuole. Alternatively, zinc may be transferred from the vacuole lumen to the ER by retrograde vesicular trafficking.
The ZRC1 gene is a Zap1 target and induced by zinc deficiency (Lyons et al., 2000; Miyabe et al., 2000). This was a surprising finding given the importance of this transporter in zinc storage and detoxification. Our previous results indicated that ZRC1 up-regulation in low zinc is required to tolerate "zinc shock" (MacDiarmid et al., 2003). Zinc shock occurs when zinc-limited cells, which express high levels of the Zrt1 zinc uptake transporter, are resupplied with zinc. The role of Zrc1 in supplying zinc to the ER is an additional reason why the ZRC1 gene may be induced in low zinc; i.e., to maintain ER zinc levels. However, given the relatively minor role Zrc1 plays in maintaining ER function (Fig. 8), our results argue that zinc shock tolerance is the major reason for the regulation of ZRC1 expression by Zap1.
We also predict that additional pathways contribute to ER zinc. UPR induction in the msc2 zrc1 cot1 triple mutant is still suppressible by adding 10 µM ZnCl2 to the medium. One possible route to bypass the loss of Msc2, Zrc1, and Cot1 activity is via fluid-phase endocytosis of zinc followed by its retrograde vesicular transport to the ER. Although formally possible, we do not favor this model based on our results. Specifically, although UPR induction in the msc2 zrc1 cot1 mutant is suppressed by 10 µM zinc, it requires 100-fold more zinc to suppress UPRE-lacZ activity in a zrt1 mutant (Fig. 1 A). Zrt1 transports zinc across the plasma membrane into the cytoplasm. Therefore, the much higher levels of zinc required to suppress UPRE-lacZ expression in a zrt1 mutant strongly argues that zinc must pass through the cytosol before entering the ER.
Msc2 is related to the Zhf protein of S. pombe (Borrelly et al., 2002; Clemens et al., 2002). Like Msc2, Zhf is a member of the CDF family of metal ion transporters. Furthermore, immunoelectron microscopy localized Zhf protein to the ER membrane. However, the phenotypic effects of zhf mutations argue that this protein plays a very different role in zinc metabolism. Although Msc2 is important for supplying zinc to the ER for organelle function, Zhf appears to be required for zinc storage and detoxification. For example, zhf mutations increase the sensitivity of cells to exogenous zinc indicating its role in detoxification. zhf mutants also have decreased zinc accumulation in cells indicating its role in zinc storage. For Msc2, we found no effect of msc2 mutations on zinc tolerance either in wild-type or zrc1 cot1 mutant cells that are greatly sensitized to exogenous zinc (unpublished data). Li and Kaplan (2000) showed that msc2 mutants actually hyperaccumulate zinc. Thus, the effects of zhf mutations in S. pombe are much more similar to mutations altering Zrc1 and Cot1 of S. cerevisiae. The ER of S. pombe may play a role in zinc storage and detoxification similar to that of the vacuole in S. cerevisiae.
Another protein related to Msc2 is mammalian ZnT-5. Although most members of the CDF family have only six transmembrane domains, Msc2 and ZnT-5 are predicted to have 15 transmembrane domains. For each, the conserved CDF region is found at the COOH terminus with several transmembrane domains attached to their NH2 termini. ZnT-5 was localized to the Golgi when expressed from the CMV promoter in HeLa cells and is widely expressed in mammalian tissues (Kambe et al., 2002). Expression was especially high in the ß cells of the pancreas in which zinc is transported into secretory vesicles for the packaging of insulin. Finally, ZnT-5dependent zinc transport activity could be observed in Golgi-derived vesicles in vitro. Given our results indicating that zinc transport into the secretory pathway is important for ER stress and UPR induction, we predict that ZnT-5 or related proteins carry out this important role in mammalian cells. Consistent with this hypothesis, we have found that expression of ZnT-5 in msc2 mutant yeast can suppress phenotypic defects of this mutant under certain conditions (unpublished data).
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Materials and methods |
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Yeast ß-galactosidase assays
ß-Galactosidase assays were performed on protein extracts (Ausubel et al., 1995) and specific activity was normalized to protein content.
Subcellular fractionation and immunoblotting
Subcellular fractionation was done largely as described by Roberg et al. (1997). Lysates were fractionated on 2060% sucrose gradients prepared with 10 mM EDTA (Mg) or with 2 mM MgSO4 (+Mg). Immunoblots were done by standard techniques (Harlow and Lane, 1988). Blots were visualized with ECL (Amersham Biosciences), and band quantitation was performed using NIH Image 1.61. Antibodies used were mouse anti-HA (12CA5, Roche), rabbit anti-HA (Sigma-Aldrich), mouse anti-Dpm1 (Molecular Probes), mouse anti-Pgk1 (Molecular Probes), goat antimouse HRP-conjugated secondary (Pierce Chemical Co.), and goat antirabbit HRP-conjugated secondary (Pierce Chemical Co.).
Assay of ERAD
Yeast were grown in 200-ml cultures to an OD600 = 0.5. Cells were harvested and resuspended in 100 ml of fresh media to a final OD600 =
1.0. Cells were grown 30 min, then cycloheximide (Sigma-Aldrich) was added to a final concentration of 100 µg/ml. 5 ml aliquots of cells were removed at each time point to tubes containing NaN3 to a final concentration of 10 mM. After the last time point, the cells were collected by centrifugation and washed once with cold buffer containing 10 mM NaN3, 1 mM EDTA. Cells were resuspended in 1 ml of the same buffer and transferred to microfuge tubes. Cells were pelleted and resuspended in 200 µl cold protein extraction buffer (10 mM Tris-Cl, pH 8, 25 mM ammonium acetate, 1 mM EDTA, 1 mM PMSF, 10% trichloroacetic acid [Sigma-Aldrich], yeast proteinase inhibitor cocktail [complete mini EDTA-free pellets; Roche]). An equal volume of glass beads was added and the tubes were vortexed five times for 1 min, with 1 min on ice between pulses. Lysates were transferred to fresh tubes. Another 500 µl of protein extraction buffer was added to the glass beads, vortexed 1 min, and then pooled with the previous lysates. Lysates were centrifuged at 14,000 g at 4°C for 10 min. The supernatant was removed and discarded. The pellets were resuspended in 120 µl buffer I (100 mM Tris base, 3% SDS, 1 mM PMSF), then boiled for 5 min. Insoluble debris was pelleted by centrifuging 5 min at 15,800 g. The supernatant was transferred to new tubes and protein concentrations were measured using the DC protein kit (Bio-Rad Laboratories). 10 µg of protein were loaded per lane when analyzed by immunoblotting.
Cell culture, transient transfection, mammalian plasmids, and assays
HeLa cells were cultured in DME (Invitrogen) plus 0.45% glucose under 5% CO2. All media contained 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 100 µM MEM nonessential amino acids (Invitrogen) supplemented with 10% FBS (Invitrogen). p5xATF6GL3 was a gift of R. Prywes (Columbia University, New York, NY), and contains five repeats of an ATF6 binding site cloned into a minimal promoter preceding the firefly luciferase gene (Wang et al., 2000). pSV-ß-galactosidase control vector (Promega) was used as a control for transfection efficiency. HeLa cells (1.2 x 106) were seeded in 60-mm plates and transiently transfected using Lipofectamine 2000 (Invitrogen). Both p5xATF6GL3 and pSV-ß-galactosidase plasmids were cotransfected in all experiments. Transfection efficiencies were typically 60%. 3648 h after transfection, tunicamycin (Sigma-Aldrich), ZnCl2, or TPEN (Sigma-Aldrich) was added to the culture media at the indicated concentrations. After treatment, the cells were washed three times with cold PBS. To generate protein extracts and perform luciferase and ß-galactosidase assays, the luciferase assay system with reporter lysis buffer (Promega) was used. Luciferase activity was normalized to ß-galactosidase activity as an internal control.
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Acknowledgments |
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This work was supported by National Institutes of Health grants GM56285 and GM69786. C.D. Ellis was supported by an MU Life Sciences Predoctoral Fellowship.
Submitted: 30 January 2004
Accepted: 9 June 2004
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1995. ß-Galactosidase assays. Current Protocols in Molecular Biology. John Wiley & Sons Inc., New York, NY. 13.6.113.6.5.
Bordallo, J., and D.H. Wolf. 1999. A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae. FEBS Lett. 448:244248.[CrossRef][Medline]
Borrelly, G.P., M.D. Harrison, A.K. Robinson, S.G. Cox, N.J. Robinson, and S.K. Whitehall. 2002. Surplus zinc is handled by Zym1 metallothionein and Zhf endoplasmic reticulum transporter in Schizosaccharomyces pombe. J. Biol. Chem. 277:3039430400.
Chang, C., and Z. Werb. 2001. The many faces of metalloproteases: cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol. 11:S37S43.[CrossRef][Medline]
Clemens, S., T. Bloss, C. Vess, D. Neumann, D.H. Nies, and U. Zur Nieden. 2002. A transporter in the endoplasmic reticulum of Schizosaccharomyces pombe cells mediates zinc storage and differentially affects transition metal tolerance. J. Biol. Chem. 277:1821518221.
Cross, F.R. 1997. "Marker swap" plasmids: convenient tools for budding yeast molecular genetics. Yeast. 13:647653.[CrossRef][Medline]
Dodson, G., and D. Steiner. 1998. The role of assembly in insulin's biosynthesis. Curr. Opin. Struct. Biol. 8:189194.[CrossRef][Medline]
Finger, A., M. Knop, and D.H. Wolf. 1993. Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. Eur. J. Biochem. 218:565574.[Abstract]
Flury, I., A. Benachour, and A. Conzelmann. 2000. YLL031c belongs to a novel family of membrane proteins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast. J. Biol. Chem. 275:2445824465.
Gaither, L.A., and D.J. Eide. 2001. Eukaryotic zinc transporters and their regulation. Biometals. 14:251270.[CrossRef][Medline]
Galperin, M.Y., and M.J. Jedrzejas. 2001. Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes. Proteins. 45:318324.[CrossRef][Medline]
Gao, C.Y., and J.L. Pinkham. 2000. Tightly regulated, beta-estradiol dose-dependent expression system for yeast. Biotechniques. 29:12261231.[Medline]
Gaynor, E.C., G. Mondesert, S.J. Grimme, S.I. Reed, P. Orlean, and S.D. Emr. 1999. MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast. Mol. Biol. Cell. 10:627648.
Gitan, R.S., H. Luo, J. Rodgers, M. Broderius, and D. Eide. 1998. Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation. J. Biol. Chem. 273:2861728624.
Grass, G., B. Fan, B.P. Rosen, S. Franke, D.H. Nies, and C. Rensing. 2001. ZitB (YbgR), a member of the cation diffusion facilitator family, is an additional zinc transporter in Escherichia coli. J. Bacteriol. 183:46644667.
Harding, H.P., M. Calfon, F. Urano, I. Novoa, and D. Ron. 2002. Transcriptional and translational control in the mammalian unfolded protein response. Annu. Rev. Cell Dev. Biol. 18:575599.[CrossRef][Medline]
Harlow, E., and D. Lane. 1988. Immunoblotting: Antibodies. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, New York. 471510.
Hinnebusch, A.G., G. Lucchini, and G.R. Fink. 1985. A synthetic HIS4 regulatory element confers general amino acid control on the cytochrome c gene (CYC1) of yeast. Proc. Natl. Acad. Sci. USA. 82:498502.[Abstract]
Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen, and L.R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 77:5159.[CrossRef][Medline]
Huang, L., C.P. Kirschke, and J. Gitschier. 2002. Functional characterization of a novel mammalian zinc transporter, ZnT6. J. Biol. Chem. 277:2638926395.
Huang, X.F., and P. Arvan. 1995. Intracellular transport of proinsulin in pancreatic ß-cells. J. Biol. Chem. 270:2041720423.
Kambe, T., H. Narita, Y. Yamaguchi-Iwai, J. Hirose, T. Amano, N. Sugiura, R. Sasaki, K. Mori, T. Iwanaga, and M. Nagao. 2002. Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells. J. Biol. Chem. 277:1904919055.
Kamizono, A., M. Nishizawa, Y. Teranishi, K. Murata, and A. Kimura. 1989. Identification of a gene conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae. Mol. Gen. Genet. 219:161167.[Medline]
Kang, T., H. Nagase, and D. Pei. 2002. Activation of membrane-type matrix metalloproteinase 3 zymogen by the proprotein convertase furin in the trans-Golgi network. Cancer Res. 62:675681.
Kawahara, T., H. Yanagi, T. Yura, and K. Mori. 1997. Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol. Biol. Cell. 8:18451862.
Kirschke, C.P., and L. Huang. 2003. ZnT7, A novel mammalian zinc transporter, accumulates zinc in the Golgi apparatus. J. Biol. Chem. 278:40964102.
Kobayashi, T., M. Beuchat, M. Lindsay, S. Frias, R.D. Palmiter, H. Sakuraba, R.G. Parton, and J. Gruenberg. 1999. Late endosomal membranes rich in lysobisphosphatidic acid regulate cholesterol transport. Nat. Cell Biol. 1:113118.[CrossRef][Medline]
Li, L., and J. Kaplan. 1997. Characterization of two homologous yeast genes that encode mitochondrial iron transporters. J. Biol. Chem. 272:2848528493.
Li, L., and J. Kaplan. 1998. Defects in the yeast high affinity iron transport system result in increased metal sensitivity because of the increased expression of transporters with a broad transition metal specificity. J. Biol. Chem. 273:2218122187.
Li, L., and J. Kaplan. 2000. The yeast gene MSC2, a member of the cation diffusion facilitator family, affects the cellular distribution of zinc. J. Biol. Chem. 276:50365043.[CrossRef][Medline]
Linke, K., T. Wolfram, J. Bussemer, and U. Jakob. 2003. The roles of the two zinc binding sites in DnaJ. J. Biol. Chem. 278:4445744466.
Lyons, T.J., A.P. Gasch, L.A. Gaither, D. Botstein, P.O. Brown, and D.J. Eide. 2000. Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast. Proc. Natl. Acad. Sci. USA. 97:79577962.
Ma, H., S. Kunes, P.J. Schatz, and D. Botstein. 1987. Plasmid construction by homologous recombination in yeast. Gene. 58:201216.[CrossRef][Medline]
MacDiarmid, C.W., L.A. Gaither, and D. Eide. 2000. Zinc transporters that regulate vacuolar zinc storage in Saccharomyces cerevisiae. EMBO J. 19:28452855.
MacDiarmid, C.W., M.A. Milanick, and D.J. Eide. 2002. Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J. Biol. Chem. 277:3918739194.
MacDiarmid, C.W., M.A. Milanick, and D.J. Eide. 2003. Induction of the ZRC1 metal tolerance gene in zinc-limited yeast confers resistance to zinc shock. J. Biol. Chem. 278:1506515072.
Mann, K.J., and D. Sevlever. 2001. 1,10-Phenanthroline inhibits glycosylphosphatidylinositol anchoring by preventing phosphoethanolamine addition to glycosylphosphatidylinositol anchor precursors. Biochemistry. 40:12051213.[CrossRef][Medline]
Miyabe, S., S. Izawa, and Y. Inoue. 2000. Expression of ZRC1 coding for suppressor of zinc toxicity is induced by zinc-starvation stress in Zap1-dependent fashion in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 276:879884.[CrossRef][Medline]
Miyabe, S., S. Izawa, and Y. Inoue. 2001. Zrc1 is involved in zinc transport system between vacuole and cytosol in Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 282:7983.[CrossRef][Medline]
Mondesert, G., D.J. Clarke, and S.I. Reed. 1997. Identification of genes controlling growth polarity in the budding yeast Saccharomyces cerevisiae: a possible role of N-glycosylation and involvement of the exocyst complex. Genetics. 147:421434.
Ng, D.T., E.D. Spear, and P. Walter. 2000. The unfolded protein response regulates multiple aspects of secretory and membrane protein biogenesis and endoplasmic reticulum quality control. J. Cell Biol. 150:7788.
Ni, L., and M. Snyder. 2001. A genomic study of the bipolar bud site selection pattern in Saccharomyces cerevisiae. Mol. Biol. Cell. 12:21472170.
Nies, D.H., and S. Silver. 1995. Ion efflux systems involved in bacterial metal resistances. J. Ind. Microbiol. 14:186199.[Medline]
Nishikawa, S., and T. Endo. 1997. The yeast JEM1p is a DnaJ-like protein of the endoplasmic reticulum membrane required for nuclear fusion. J. Biol. Chem. 272:1288912892.
Nishikawa, S.I., S.W. Fewell, Y. Kato, J.L. Brodsky, and T. Endo. 2001. Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation. J. Cell Biol. 153:10611070.
Palmiter, R.D., and S.D. Findley. 1995. Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO J. 14:639649.[Abstract]
Palmiter, R.D., and L. Huang. 2004. Efflux and compartmentalization of zinc by members of the SLC30 family of solute carriers. Pflugers Arch. 447:744751.[CrossRef][Medline]
Palmiter, R.D., T.B. Cole, and S.D. Findley. 1996. ZnT-2, a mammalian protein that confers resistance to zinc by facilitating vesicular sequestration. EMBO J. 15:17841791.[Abstract]
Patil, C., and P. Walter. 2001. Intracellular signaling from the endoplasmic reticulum to the nucleus: the unfolded protein response in yeast and mammals. Curr. Opin. Cell Biol. 13:349355.[CrossRef][Medline]
Paulsen, I.T., and M.H. Saier. 1997. A novel family of ubiquitous heavy metal ion transport proteins. J. Membr. Biol. 156:99103.[CrossRef][Medline]
Pei, D., and S.J. Weiss. 1995. Furin-dependent intracellular activation of the human stromelysin-3 zymogen. Nature. 375:244247.[CrossRef][Medline]
Roberg, K.J., N. Rowley, and C.A. Kaiser. 1997. Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae. J. Cell Biol. 137:14691482.
Sevlever, D., K.J. Mann, and M.E. Medof. 2001. Differential effect of 1,10-phenanthroline on mammalian, yeast, and parasite glycosylphosphatidylinositol anchor synthesis. Biochem. Biophys. Res. Commun. 288:11121118.[CrossRef][Medline]
Shulga, N., N. Mosammaparast, R. Wozniak, and D.S. Goldfarb. 2000. Yeast nucleoporins involved in passive nuclear envelope permeability. J. Cell Biol. 149:10271038.
Silberstein, S., G. Schlenstedt, P.A. Silver, and R. Gilmore. 1998. A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum. J. Cell Biol. 143:921933.
Swanson, R., M. Locher, and M. Hochstrasser. 2001. A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Mat2 repressor degradation. Genes Dev. 15:26602674.
Tang, W., and C. Wang. 2001. Zinc fingers and thiol-disulfide oxidoreductase activities of chaperone DnaJ. Biochemistry. 40:1498514994.[CrossRef][Medline]
Taron, C.H., J.M. Wiedman, S.J. Grimme, and P. Orlean. 2000. Glycosylphosphatidylinositol biosynthesis defects in Gpi11p- and Gpi13p-deficient yeast suggest a branched pathway and implicate gpi13p in phosphoethanolamine transfer to the third mannose. Mol. Biol. Cell. 11:16111630.
Thompson, D.A., and F.W. Stahl. 1999. Genetic control of recombination partner preference in yeast meiosis: isolation and characterization of mutants elevated for meiotic unequal sister-chromatid recombination. Genetics. 153:621641.
Tohe, A., and T. Oguchi. 1999. Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae. Genes Genet. Syst. 74:241256.[CrossRef][Medline]
Travers, K.J., C.K. Patil, L. Wodicka, D.J. Lockhart, J.S. Weissman, and P. Walter. 2000. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 101:249258.[Medline]
Wang, Y., J. Shen, N. Arenzana, W. Tirasophon, R.J. Kaufman, and R. Prywes. 2000. Activation of ATF6 and an ATF6 DNA binding site by the endoplasmic reticulum stress response. J. Biol. Chem. 275:2701327020.
Waters, B.M., and D.J. Eide. 2002. Combinatorial control of yeast FET4 gene expression in response to iron, zinc, and oxygen. J. Biol. Chem. 277:3374933757.
Zhao, H., and D. Eide. 1996a. The yeast ZRT1 gene encodes the zinc transporter of a high affinity uptake system induced by zinc limitation. Proc. Natl. Acad. Sci. USA. 93:24542458.
Zhao, H., and D. Eide. 1996b. The ZRT2 gene encodes the low affinity zinc transporter in Saccharomyces cerevisiae. J. Biol. Chem. 271:2320323210.
Zhao, H., and D.J. Eide. 1997. Zap1p, a metalloregulatory protein involved in zinc-responsive transcriptional regulation in Saccharomyces cerevisiae. Mol. Cell. Biol. 17:50445052.[Abstract]
Zhao, H., E. Butler, J. Rodgers, T. Spizzo, and S. Duesterhoeft. 1998. Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements. J. Biol. Chem. 273:2871328720.