FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2073195145> ?p ?o ?g. }

• W2073195145 endingPage "33394" @default.
• W2073195145 startingPage "33388" @default.
• W2073195145 abstract "Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis. The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles. In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes. Each null mutation exhibited sensitivity to metal ion chelators at different concentrations. The combination of null mutants ΔSMF1 + ΔSMF2and the triple null mutant Δ3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively. Addition of 5 μmcopper or 25 μm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent. The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein. Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Δ3SMF. Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes. The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3and FET4 were inactivated. Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium. We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells. Metal ions are vital for all organisms, and metal ion transporters play a crucial role in maintaining their homeostasis. The yeast (Saccharomyces cerevisiae) Smf transporters and their homologs in other organisms have a central role in the accumulation of metal ions and their distribution in different tissues and cellular organelles. In this work we generated null mutations in each individual SMF gene in yeast as well as in all combinations of the genes. Each null mutation exhibited sensitivity to metal ion chelators at different concentrations. The combination of null mutants ΔSMF1 + ΔSMF2and the triple null mutant Δ3SMF failed to grow on medium buffered at pH 8 and 7.5, respectively. Addition of 5 μmcopper or 25 μm manganese alleviated the growth arrest at the high pH or in the presence of the chelating agent. The transport of manganese was analyzed in the triple null mutant and in this mutant expressing each Smf protein. Although overexpression of Smf1p and Smf2p resulted in uptake that was higher than wild type cells, the expression of Smf3p gave no significant uptake above that of the triple mutant Δ3SMF. Western analysis with antibody against Smf3p indicated that this transporter does not reach the plasma membrane and may function at the Golgi or post-Golgi complexes. The iron uptake resulting from expression of Smf1p and Smf2p was analyzed in a mutant in which its iron transporters FET3and FET4 were inactivated. Overexpression of Smf1p gave rise to a significant iron uptake that was sensitive to the sodium concentrations in the medium. We conclude that the Smf proteins play a major role in copper and manganese homeostasis and, under certain circumstances, Smf1p may function in iron transport into the cells. 4-morpholineethanesulfonic acid kilobase(s) polymerase chain reaction 4-morpholinepropanesulfonic acid transmembrane domain target SNAP receptor Transition metals are essential for many metabolic processes, and their homeostasis is crucial for life processes. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast (Saccharomyces cerevisiae) mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs (1Zhou B. Gitschier J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7481-7486Crossref PubMed Scopus (474) Google Scholar, 2Hung I.H. Suzuki M. Yamaguchi Y. Yuan D.S. Klausner R.D. Gitlin J.D. J. Biol. Chem. 1997; 272: 21461-21466Abstract Full Text Full Text PDF PubMed Scopus (287) Google Scholar, 3Csere P. Lill R. Kispal G. FEBS Lett. 1998; 441: 266-270Crossref PubMed Scopus (120) Google Scholar, 4Tabuchi M. Yoshida T. Takegawa K. Kishi F. Biochem. J. 1999; 344: 211-219Crossref PubMed Google Scholar). Some yeast and human homologous proteins were found to be related to copper and iron transport (5Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (244) Google Scholar, 6Andrews N.C. Levy J.E. Blood. 1998; 92: 1845-1851Crossref PubMed Google Scholar, 7Radisky D.C. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), among which the most ubiquitous are theSMF family of genes encoding metal ion transporters (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar, 9Supek F. Supekova L. Nelson H. Nelson N. J. Exp. Biol. 1997; 200: 321-330PubMed Google Scholar, 10Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (253) Google Scholar).SMF1 was originally cloned as a high copy number suppressor of a temperature-sensitive mif1-1 mutant (11West A.H. Clark D.J. Martin J. Neupert W. Hartl F.-U. Horwich A.L. J. Biol. Chem. 1992; 267: 24625-24633Abstract Full Text PDF PubMed Google Scholar). Later it was shown that the growth arrest at 37 °C could be relieved by supplementing the media with Mn2+ or overexpressingSMF1 that transports Mn2+ from the medium and elevates its concentration in the cytoplasm (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar, 12Liu X.F. Supek F. Nelson N. Culotta V.C. J. Biol. Chem. 1997; 272: 11763-11769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 13Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The temperature-sensitive mif1-1 mutant may have resulted from reduced stability of the processing peptidase under limited manganese concentrations in the medium (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar). Further studies indicated theSMF1 is a general metal ion transporter and can transport not only Mn2+, Zn2+, and Cu2+ (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar), but also Fe2+, Cd2+, Ni2+, and Co2+ (9Supek F. Supekova L. Nelson H. Nelson N. J. Exp. Biol. 1997; 200: 321-330PubMed Google Scholar, 12Liu X.F. Supek F. Nelson N. Culotta V.C. J. Biol. Chem. 1997; 272: 11763-11769Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 14Chen X.-Z. Peng J.-B. Cohen A. Nelson H. Nelson N. Hediger M.A. J. Biol. Chem. 1999; 274: 35089-35094Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Yeast cells contain additional two genes of this family, SMF2 and SMF3, and indirect evidence indicates that they are also broad range metal ion transporters but exhibit different specificity from SMF1(10Nelson N. EMBO J. 1999; 18: 4361-4371Crossref PubMed Scopus (253) Google Scholar), suggesting a specific function for each of them. Expression of Smf1p in Xenopus oocytes demonstrated that this protein mediates H+-dependent divalent metal ion transport (14Chen X.-Z. Peng J.-B. Cohen A. Nelson H. Nelson N. Hediger M.A. J. Biol. Chem. 1999; 274: 35089-35094Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In addition, a large Na+ leak through Smf1p was observed, and sodium competed with the activity of metal ion uptake. Because the Smf family of proteins transports a wide range of divalent metal ions and specific and highly regulated transport systems exist in yeast for several of those metals (5Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (244) Google Scholar, 6Andrews N.C. Levy J.E. Blood. 1998; 92: 1845-1851Crossref PubMed Google Scholar, 7Radisky D.C. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the function of the Smf family of proteins is not apparent. Our approach, since the discovery of these family members as metal ion transporters (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar), was to identify conditions and yeast mutants that will help to elucidate specific functions of the various genes. Here we report on the properties of deletion mutants in each of the genes encoding SMF1, SMF2, and SMF3, as well as the combination of multiple deletions, including a triple mutant lacking the three genes. Our study revealed that the Smf proteins take part in the transport of Cu2+ and Mn2+ into yeast cells, and in their absence a growth arrest occurs due to a shortage in these metal ions. The “wild-type” that was used is S. cerevisiae W303 (MATa/α trp1 ade2 his3 leu2 ura3). The other strains used in this work are: ΔSMF1(MATα ade2 his3 leu2 trp1 SMF1::URA3); ΔSMF2(MATα ade2 his3 trp1 leu2 SMF2::URA3); ΔSMF3(MATα ade2 his3 trp1 leu2 SMF3::URA3); ΔSMF1+2(MATα ade2 his3 leu2 ura3 SMF1::URA3/FOA SMF2::URA3); ΔSMF1+3(MATα ade2 his3 leu2 ura3 SMF1::URA3/FOA SMF3::URA3); ΔSMF2+3(MATα ade2 his3 leu2 ura3 SMF2::URA3/FOA SMF3:: URA3);ΔSMF1+2+3 (MATα ade2 his3 leu2 ura3 SMF1::URA3/FOA SMF2::URA3/FOA SMF3::URA3). The yeast strain in which FET3 and FET4 genes were inactivated (Δ2FET) was the DEY 1453 (15Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1067) Google Scholar). The cells were grown in a YPD medium containing 1% yeast extract, 2% bactopeptone, and 2% dextrose. For metal ion limitation experiments, the cells were grown in a medium containing 0.25% yeast extract, 0.5% bactopeptone 2% dextrose, 50 mmMES,1 and the pH was usually adjusted to pH 6 by NaOH (16Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3503-3507Crossref PubMed Scopus (245) Google Scholar, 17Noumi T. Beltrán C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1938-1942Crossref PubMed Scopus (128) Google Scholar). Agar plates were prepared by the addition of 2% agar to the YPD buffer medium at the given pH. Yeast transformation was performed as described previously (18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar), and the transformed cells were grown on minimal plates containing a 0.67% yeast nitrogen base, 2% dextrose, 2% agar, and the appropriate nutritional requirements. The gene knockout of the new strains was performed as follows: All or part of the target gene was replaced by the selectable marker URA3, leaving flanking DNA sequences of about 0.3 kb. When PCR was used for the construct, the DNA fragments were cloned into the TA plasmid or pGEM-T Easy (Promega). Transformed colonies that grew on the selective medium were selected, checked by PCR for homologous recombination, and analyzed for their phenotype. The genes containing approximately 0.3-kb flanking sequences were cloned by PCR into YEP24 or YPN2 or BFG plasmids (17Noumi T. Beltrán C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1938-1942Crossref PubMed Scopus (128) Google Scholar). Sequential gene disruption was obtained by inactivation of the URA3 gene and selection on minimal plates containing 1 mg/ml 5-fluorouracil. The colonies that grew under this condition were analyzed for lack of growth on minimal plates without uracil. These yeast strains were used for subsequent gene disruption with a URA3-selectable marker. The various null mutants were analyzed for the disruption of each gene by PCR using one primer from the URA3 gene and one primer flanking the interrupted gene. The interruption of SMF1 was performed as described previously (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar). SMF2 was interrupted by the PCR-only (HANNAH) method (19Supekova L. Supek F. Nelson N. J. Biol. Chem. 1995; 270: 13726-13732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The 5′- and 3′-flanking regions of the gene, as well as the selectable marker, were amplified by PCR using oligonucleotides that are partially complementary to yield, in a second PCR, a DNA fragment composed of 5′-SMF2 URA3 3′-SMF2. Most of the reading frame of the gene was deleted leaving DNA fragments encoding 14 and 18 amino acids at the N and C termini of the transporter, respectively. SMF3 was interrupted by introducing the URA3 gene in the StyI site ofSMF3. The same constructs were used for sequential interruption of more than one SMF gene. Yeast transformation was performed either by the method of Ito et al. (18Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar) or by a bench-top method according to Elble (20Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar). Yeast cells were grown overnight in 5 ml of YPD medium (pH 5.5) to stationary phase. The cells were centrifuged for 10 s in an Eppendorf microcentrifuge at 13,000 rpm. 10 μl of salmon sperm (10 mg/ml) was added to the pellet as a DNA carrier. Then about 1 μg of plasmid or DNA construct was added. Finally, the pellet was suspended in 0.5 ml of medium containing 10 mm Tris, pH 7.5, 1 mm EDTA, 40% polyethylene glycol 4000, and 0.1 m lithium acetate. The suspension was incubated overnight at room temperature and plated on the appropriate plates (17Noumi T. Beltrán C. Nelson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1938-1942Crossref PubMed Scopus (128) Google Scholar). Yeast cells were grown in 5 ml of selective or YPD medium to stationary phase. The cells were harvested by centrifugation for 2 min at 2500 rpm. The pellet was suspended in 100 μl of STET solution containing 50 mm Tris (pH 8), 50 mm EDTA, 5% Triton X-100, and 8% sucrose. Glass beads (about 0.2 g) were added, and the suspension was vortexed for 20 min. Then, an additional 100 μl of STET solution was added, and the mixture was boiled for 3 min, cooled for 1 min on ice, and centrifuged for 10 min at 18,000 × g. 100 μl was removed from the supernatant and 50 μl of 7.5 m ammonium acetate was added. The mixture was incubated for 1 h at −20 °C and centrifuged for 10 min at 18,000 × g. 100 μl of supernatant was removed to a fresh tube, 200 μl of cold ethanol was added, and the mixture was centrifuged for 30 min at 18,000 ×g. The pellet, containing the DNA, was washed with 70% ethanol and dissolved in 20 μl of 10 mm Tris and 1 mm EDTA (pH 8). Polyclonal antibody against Smf3p was obtained by injecting rabbits with a chimeric protein containing the maltose-binding protein and the hydrophilic sequence of amino acids 382–469 of the Smf3p. The DNA fragment encoding these amino acids was amplified by PCR with introduced EcoRI and HindIII restriction sites. The amplified DNA fragment was cloned in-frame to the maltose binding protein in the plasmid PMAL-C (New England BioLabs). Following sequence verification, 500 ml of bacterial culture was grown to OD 0.5 at 600 nm, induced with isopropyl-1-thio-β-d-galactopyranoside for 3 h, and harvested by centrifugation at 4000 ×g. The cells were disrupted by French press, and the protein was purified by using a column containing maltose agarose. The fractions containing the chimeric protein were dissociated by SDS, loaded on preparative gel, and electrophoresed. The gel was briefly stained by Coomassie Blue, the identified protein band was cut out, and the fusion protein was electroeluted. About 0.25 mg of fusion protein was injected into rabbits as described previously (21Nelson N. Methods Enzymol. 1983; 97: 510-523Crossref Scopus (31) Google Scholar). Antibody to Pma1p was raised in rabbits using the purified protein that was electroeluted from polyacrylamide gels as described previously (21Nelson N. Methods Enzymol. 1983; 97: 510-523Crossref Scopus (31) Google Scholar, 22Koland J.G. Hammes G.G. J. Biol. Chem. 1986; 261: 5936-5942Abstract Full Text PDF PubMed Google Scholar, 23Cohen A. Perzov N. Nelson H. Nelson N. J. Biol. Chem. 1999; 274: 26885-26893Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Antibody against Sed5p was a generous gift from Dr. Randy Schekman (University of California, Berkeley, CA). The antibody detection system (ECL) was from Amersham Pharmacia Biotech. Western blots were performed according to the protocol of the ECL antibody detection system from the manufacturer. Samples were denatured by SDS sample buffer and electrophoresed on 12% polyacrylamide Mini gels (Bio-Rad) as described previously (24Nelson H. Mandiyan S. Nelson N. J. Biol. Chem. 1994; 269: 24150-24155Abstract Full Text PDF PubMed Google Scholar). Following electrotransfer at 0.5 A for 15 min, the nitrocellulose filters were blocked for 1 h in a solution containing 100 mm NaCl, 100 mm sodium phosphate (pH 7.5), 0.1% Tween 20, and 5% nonfat dried milk. Antibodies were incubated for 30 min at room temperature at a dilution of 1:1000 in a similar solution containing 2% dried milk. Following five washes in the same solution, peroxidase-conjugated second antibody or protein A was added to the filters. After incubation for 30 min and five washes with the same solution, the nitrocellulose filters were subjected to the ECL amplification procedure. The filters were exposed to Kodak X-Omat AR film for 5–60 s. Yeast cells were grown in 500 ml of YPD medium (pH 5.5) to OD 1 at 600 nm. The suspension was centrifuged at 3000 × g for 5 min, and the pellet was washed with 200 ml of water and then washed again with 1 msorbitol. The cell wall was digested by 2.5 units of zymolyase in 10 ml of solution containing 10 mm HEPES, pH 7.5, and 1m sorbitol. After 30-min incubation at 30 °C, the suspension was centrifuged in 15-ml Corex tubes at 3000 ×g for 5 min. 1 ml of glass beads was added to the pellet as well as 1 ml of solution containing 30 mm MOPS (pH 7), 1:100 protease inhibitor mixture (Sigma), 1 mmphenylmethylsulfonyl fluoride, 1 mm EDTA, and 1 mm EGTA. The suspension was vortexed five times for 30 s with incubation on ice for 30 s between each vortexing step. The solution was removed from the glass beads and placed in a new Corex tube. An additional 2 ml of the above solution was added to the tube with the glass beads, and the tube was vortexed briefly. The suspension was added to the previous one and centrifuged at 1000 ×g for 5 min to give a pellet containing the cell debris and nuclei. The supernatant was centrifuged at 12,000 × gfor 10 min, and the pellet was suspended in 0.3–0.5 ml of a solution containing 10 mm Hepes (pH 7.5) and 0.5 msorbitol and stored as the mitochondrial fraction. The supernatant was centrifuged at 115,000 × g for 30 min, and the pellet was suspended in 0.3–0.5 ml of a solution containing 10 mmTris-Cl (pH 7.5), 1 mm EDTA, 2 mmdithiothreitol, and 25% glycerol and stored as the membrane fraction at −80 °C. Sucrose gradients were also used to estimate the relative density of various membrane fractions. The gradients were made as described in Lupashin et al. (25Lupashin V.V. Pokrovskaya I.D. McNew J. Waters M.G. Mol. Biol. Cell. 1997; 8: 2659-2676Crossref PubMed Scopus (92) Google Scholar), except that gradients of 20–60% sucrose were used and the centrifugation was done for 14 h. SMF1 encodes a hydrophobic protein of 63,258 Da with potentially eight to ten transmembrane domains. A search in the yeast genome data base with the Smf1p sequence revealed two homologous genes that were namedSMF2 and SMF3. These genes encode proteins of 59,758 Da and 51,778 Da, respectively, and contain a similar number of transmembrane domains. Fig. 1 shows the multiple alignment of the predicted amino acid sequences of the three members of the yeast SMF gene family. The three proteins exhibit about 50% identity to each other, and the main difference between them was found at their N terminus. At this end, Smf3p is shorter then Smf1p by 70 amino acids and by 51 amino acids from Smf2p. These extra pieces are highly populated by charged amino acids. Up to 12 transmembrane segments have been proposed to constitute DCT1, the mammalian homolog of Smf1p (26Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2668) Google Scholar). We assume that the number of transmembrane segments will be similar in all the family members and 10 or 12 transmembrane segments are likely to exist in these transporters. Multiple alignment of amino acid sequences of family members from bacteria, yeast, plants, insects, and mammals suggests that several conserved charged amino acids are present inside the membrane. Among them (using Smf1p amino acid sequence) are Asp-92 in TM1; Glu-160, Asp-167, and Glu-170 in TM3; and His-278 in TM6. The three negatively charged amino acids in TM3 face the same side of an α helix, suggesting a role in translocation of positively charged ions across the membrane. Obviously, the above structural and functional assumptions have to be examined by multiple experimental approaches.Figure 2Effect of EGTA on growth of the various combinations of SMF disruptant mutants. The buffered YPD plates (pH 6) were prepared as described under “Materials and Methods.” The indicated concentrations of filter-sterile sodium-EGTA were added right before the pouring of the warm medium. Cultures of the various yeast strains were washed by sterile distilled water and 5 ml of the cell suspension, adjusted to 0.001 OD, were placed in the indicated positions. The plates were incubated for 2 days at 30 °C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1Multiple alignment of the amino acid sequences of the SMF family members. The amino acid sequences of Smf1p through Smf3p were obtained from GenBank™ as the following reading frames: Smf1p,YOL122c; Smf2p, YHR050w; and Smf3p, YLR034c. The multiple alignment was done using the program Pileup. The Boxshade program was used for visualizing the results (gcg software package).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The main feature of the SMF1 null mutant was its sensitivity to EGTA concentrations, toward which the parental wild-type strain was insensitive (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar). The growth inhibition of ΔSMF1in the presence of EGTA could be alleviated by low concentrations of manganese or copper but not by any other metal ion. Deletion of the genes encoding SMF1 and SMF2 resulted in a double null mutant that was not able to grow at pH 8 but exhibited normal growth in YPD medium buffered at lower pH (27Pinner E. Gruenheid S. Raymond M. Gros P. J. Biol. Chem. 1997; 272: 28933-28938Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). We generated null mutations in each individual SMF gene as well as combinations of null mutations in these genes. The triple null mutant in the three SMF genes (Δ3SMF) was highly sensitive to EGTA and was not able to grow on YPD medium buffered at pH 7.5. Table Isummarizes the sensitivity and resistance of the various null mutants to the chelator EGTA, pH, various metal ions, high osmolarity, and oxidative stress (obtained by the addition of H2O2 to the medium). All the nullSMF mutants exhibited some sensitivity to EGTA. AlthoughΔSMF1 and ΔSMF2 were quite sensitive to EGTA, ΔSMF3 showed only marginal sensitivity to the chelating agent. The mutant strainsΔSMF1, ΔSMF3, andΔSMF1+3 exhibited resistance to relatively high Co2+ and Mn2+ concentrations in the medium.ΔSMF2 showed resistance to osmotic stress induced by 0.9 m NaCl or 1.6 m glycerol or 1.5 or 1.7 m sorbitol, as well as relative resistance to Mn2+, but only at pH 7.5. The resistance for certain metal ions can be explained in terms of reduced transport activity of these ions in the absence of one or more of the Smf metal ion transporters. It is more difficult to account for the sensitivity to metal ions in the various null mutants. Thus ΔSMF2 mutant was sensitive to Zn2+ and Ni2+;ΔSMF1+2 was sensitive to Ni2+, Co2+, and Mn2+; andΔSMF1+3 was sensitive to Zn2+. Apparently, disturbances in metal ion homeostasis may elicit pleiotropic effects through alteration and different distribution of the other metal ion transporters and/or signal transduction mediators (7Radisky D.C. Kaplan J. J. Biol. Chem. 1999; 274: 4481-4484Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Supek F. Supekova L. Nelson H. Nelson N. J. Exp. Biol. 1997; 200: 321-330PubMed Google Scholar, 13Liu X.F. Culotta V.C. J. Biol. Chem. 1999; 274: 4863-4868Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 28Ooi C.E. Rabinovich E. Dancis A. Bonifacino J.S. Klausner R.D. EMBO J. 1996; 15: 3515-3523Crossref PubMed Scopus (180) Google Scholar, 29Yuan D.S. Dancis A. Klausner R.D. J. Biol. Chem. 1997; 272: 25787-25793Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Askwith C. Kaplan J. Trends Biochem. Sci. 1998; 23: 135-138Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 31Li L. Kaplan J. J. Biol. Chem. 1998; 273: 22181-22187Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Table IPhenotypes resulted from inactivation of each SMF gene and all the combination of SMF null mutationsMutantSensitivityResistanceΔSMF1EGTACo2+, Mn2+ΔSMF2EGTA, Zn2+, Ni2+NaCl, sorbitol, H2O2ΔSMF3EGTACo2+, Mn2+ΔSMF1+2EGTA, Ni2+, Co2+, Mn2+ΔSMF1+3EGTA, Zn2+Co2+, Mn2+ΔSMF2+3EGTAΔSMF1+2+3 (Δ3SMF)EGTA, H2O2, pH 7.5, Zn2+The assay for resistance and sensitivity for metal ions was performed on a solid YPD medium buffered at pH 6 or the indicated pH. The medium was supplemented with the indicated metal ions in their chloride form at the following concentrations: Co2+, 0.25, 0.5, 0.75 mm; Mn2+, 1.5, 2, 2.5 mm; Zn2+, 5, 6, 7 mm; Ni2+, 1.5, 2, 2.5 mm. Sensitivity to EGTA was assayed as described in Fig. 2. Resistance to NaCl (0.9 m) and sorbitol (1.5 or 1.7 m) was analyzed at pH 6. Sensitivity or resistance to H2O2 was measured in liquid medium buffered at pH 6 at concentrations of 0.5 and 2 mm. Cells were inoculated to give a cell density of 0.2 OD at 600 nm, and the growth rate was measured every 2 h by following the increase in optical absorption at the same wavelength. Open table in a new tab The assay for resistance and sensitivity for metal ions was performed on a solid YPD medium buffered at pH 6 or the indicated pH. The medium was supplemented with the indicated metal ions in their chloride form at the following concentrations: Co2+, 0.25, 0.5, 0.75 mm; Mn2+, 1.5, 2, 2.5 mm; Zn2+, 5, 6, 7 mm; Ni2+, 1.5, 2, 2.5 mm. Sensitivity to EGTA was assayed as described in Fig. 2. Resistance to NaCl (0.9 m) and sorbitol (1.5 or 1.7 m) was analyzed at pH 6. Sensitivity or resistance to H2O2 was measured in liquid medium buffered at pH 6 at concentrations of 0.5 and 2 mm. Cells were inoculated to give a cell density of 0.2 OD at 600 nm, and the growth rate was measured every 2 h by following the increase in optical absorption at the same wavelength. One of the key questions to be answered is how do seemingly unrelated metal ions elicit similar effect in mutants lacking one or more of theSMF genes? Inactivation of the Drosophila homolog of the SMF1 gene resulted in a loss of taste behavior (32Rodrigues V. Cheah P.Y. Ray K. Chia W. EMBO J. 1995; 14: 3007-3020Crossref PubMed Scopus (126) Google Scholar). Addition of manganese or iron to the food of these mutants corrected the defect in their taste behavior (33Orgad S. Nelson H. Segal D. Nelson N. J. Exp. Biol. 1998; 201: 115-120Crossref PubMed Google Scholar). Similarly, addition of micromolar concentrations of Cu2+ or Mn2+relieved the growth arrest of the yeast mutant in which theSMF1 gene was interrupted (8Supek F. Supekova L. Nerlson H. Nelson N. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5105-5110Crossref PubMed Scopus (286) Google Scholar). To gain further information on the phenotype of the various SMF null mutants, we tested their growth under different EGTA concentrations. Fig.2 shows a sensitivity order toward EGTA of ΔSMF1 >ΔSMF2 > ΔSMF3 for the respective null mutants. The parental wild-type strain grew quite well at EGTA concentrations of up to 3 mm. Reduced growth was already detected at 0.5 mm EGTA for the mutantsΔSMF1 and ΔSMF2, and a complete growth arrest of these mutants was obtained at 2 mm EGTA. The mutant ΔSMF3 grew well on plates containing 1 mm EGTA and a complete growth arrest of this mutant was obtained only at 3 mm EGTA. The triple mutant ΔSMF1+2+3 was highly sensitive to the chelating agent and also exhibited pH sensitivity. It grew normally at pH 5.5, grew poorly at pH 6.5, and exhibited a complete growth arrest at pH 7.5 (Fig. 4). 2A. Cohen" @default.
• W2073195145 created "2016-06-24" @default.
• W2073195145 creator A5023301230 @default.
• W2073195145 creator A5054296185 @default.
• W2073195145 creator A5085637404 @default.
• W2073195145 date "2000-10-01" @default.
• W2073195145 modified "2023-09-26" @default.
• W2073195145 title "The Family of SMF Metal Ion Transporters in Yeast Cells" @default.
• W2073195145 cites W119688228 @default.
• W2073195145 cites W1480048429 @default.
• W2073195145 cites W1514437369 @default.
• W2073195145 cites W1535147323 @default.
• W2073195145 cites W1568628619 @default.
• W2073195145 cites W1599576575 @default.
• W2073195145 cites W1696228719 @default.
• W2073195145 cites W1963874808 @default.
• W2073195145 cites W1973782344 @default.
• W2073195145 cites W1975190250 @default.
• W2073195145 cites W1976104570 @default.
• W2073195145 cites W1994710858 @default.
• W2073195145 cites W1997682893 @default.
• W2073195145 cites W2007049169 @default.
• W2073195145 cites W2007632640 @default.
• W2073195145 cites W2010355719 @default.
• W2073195145 cites W2015326313 @default.
• W2073195145 cites W2016067359 @default.
• W2073195145 cites W2022828571 @default.
• W2073195145 cites W2025144653 @default.
• W2073195145 cites W2038142268 @default.
• W2073195145 cites W2038464932 @default.
• W2073195145 cites W204545715 @default.
• W2073195145 cites W2046818599 @default.
• W2073195145 cites W2047023472 @default.
• W2073195145 cites W2047390883 @default.
• W2073195145 cites W2070330324 @default.
• W2073195145 cites W2074278834 @default.
• W2073195145 cites W2087276505 @default.
• W2073195145 cites W2088152932 @default.
• W2073195145 cites W2090401018 @default.
• W2073195145 cites W2097888227 @default.
• W2073195145 cites W2104786801 @default.
• W2073195145 cites W2105568529 @default.
• W2073195145 cites W2106442871 @default.
• W2073195145 cites W2112169138 @default.
• W2073195145 cites W2119294007 @default.
• W2073195145 cites W2123982967 @default.
• W2073195145 cites W2137964854 @default.
• W2073195145 cites W261351262 @default.
• W2073195145 cites W4250625093 @default.
• W2073195145 cites W43717001 @default.
• W2073195145 cites W967598526 @default.
• W2073195145 doi "https://doi.org/10.1074/jbc.m004611200" @default.
• W2073195145 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10930410" @default.
• W2073195145 hasPublicationYear "2000" @default.
• W2073195145 type Work @default.
• W2073195145 sameAs 2073195145 @default.
• W2073195145 citedByCount "131" @default.
• W2073195145 countsByYear W20731951452012 @default.
• W2073195145 countsByYear W20731951452013 @default.
• W2073195145 countsByYear W20731951452014 @default.
• W2073195145 countsByYear W20731951452015 @default.
• W2073195145 countsByYear W20731951452016 @default.
• W2073195145 countsByYear W20731951452017 @default.
• W2073195145 countsByYear W20731951452018 @default.
• W2073195145 countsByYear W20731951452019 @default.
• W2073195145 countsByYear W20731951452020 @default.
• W2073195145 countsByYear W20731951452021 @default.
• W2073195145 countsByYear W20731951452022 @default.
• W2073195145 countsByYear W20731951452023 @default.
• W2073195145 crossrefType "journal-article" @default.
• W2073195145 hasAuthorship W2073195145A5023301230 @default.
• W2073195145 hasAuthorship W2073195145A5054296185 @default.
• W2073195145 hasAuthorship W2073195145A5085637404 @default.
• W2073195145 hasBestOaLocation W20731951451 @default.
• W2073195145 hasConcept C104317684 @default.
• W2073195145 hasConcept C149011108 @default.
• W2073195145 hasConcept C178790620 @default.
• W2073195145 hasConcept C185592680 @default.
• W2073195145 hasConcept C2779222958 @default.
• W2073195145 hasConcept C544153396 @default.
• W2073195145 hasConcept C55493867 @default.
• W2073195145 hasConcept C86803240 @default.
• W2073195145 hasConcept C95444343 @default.
• W2073195145 hasConceptScore W2073195145C104317684 @default.
• W2073195145 hasConceptScore W2073195145C149011108 @default.
• W2073195145 hasConceptScore W2073195145C178790620 @default.
• W2073195145 hasConceptScore W2073195145C185592680 @default.
• W2073195145 hasConceptScore W2073195145C2779222958 @default.
• W2073195145 hasConceptScore W2073195145C544153396 @default.
• W2073195145 hasConceptScore W2073195145C55493867 @default.
• W2073195145 hasConceptScore W2073195145C86803240 @default.
• W2073195145 hasConceptScore W2073195145C95444343 @default.
• W2073195145 hasIssue "43" @default.
• W2073195145 hasLocation W20731951451 @default.
• W2073195145 hasOpenAccess W2073195145 @default.
• W2073195145 hasPrimaryLocation W20731951451 @default.
• W2073195145 hasRelatedWork W1274460528 @default.
• W2073195145 hasRelatedWork W1895811 @default.

• next