Copper inhibits protein maturation in the secretory pathway by targeting the Sec61 translocon in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum. These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the endoplasmic reticulum. Here, using immunoblotting, microscopy, and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated proteins, Gas1 (glycophospholipid-anchored surface protein) and CPY (carboxypeptidase Y), in the cytosol. We further show that the copper-sensitive phenotype of sec61-deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Furthermore, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper-mediated accumulation of post-translationally translocated proteins is due to the inhibition of Sec61. In Saccharomyces cerevisiae, proteins destined for secretion utilize the post-translational translocon machinery to gain entry into the endoplasmic reticulum. These proteins then mature by undergoing a number of post-translational modifications in different compartments of the secretory pathway. While these modifications have been well established for many proteins, to date only a few studies have been conducted regarding the conditions and factors affecting maturation of these proteins before entering into the endoplasmic reticulum. Here, using immunoblotting, microscopy, and spot test assays, we show that excess copper inhibits the Sec61 translocon function and causes accumulation of two well-known post-translationally translocated proteins, Gas1 (glycophospholipid-anchored surface protein) and CPY (carboxypeptidase Y), in the cytosol. We further show that the copper-sensitive phenotype of sec61-deficient yeast cells is ameliorated by restoring the levels of SEC61 through plasmid transformation. Furthermore, screening of translocation-defective Sec61 mutants revealed that sec61-22, bearing L80M, V134I, M248V, and L342S mutations, is resistant to copper, suggesting that copper might be inflicting toxicity through one of these residues. In conclusion, these findings imply that copper-mediated accumulation of post-translationally translocated proteins is due to the inhibition of Sec61. Copper, an essential element, is required by multiple enzymes, such as lysyl oxidase, cytochrome C, superoxide dismutase, and others (1Kodama H. Fujisawa C. Bhadhprasit W. Inherited copper transport disorders: Biochemical mechanisms, diagnosis, and treatment.Curr. Drug Metab. 2012; 13: 237-250Google Scholar). Copper sensing in yeast and ergo, the decision for its uptake, or sequestration/export are mediated primarily by two transcription factors, Ace1 and Mac1 (2Keller G. Bird A. Winge D.R. Independent metalloregulation of Ace1 and Mac1 in Saccharomyces cerevisiae.Eukaryot. Cell. 2005; 4: 1863-1871Google Scholar). If the cells sense excessive copper in the extracellular milieu, then Ace1 undergoes a change in its conformation to convert into an active form. This activated transcription factor binds to the promoter of genes like CUP1, CRS5 (metallothioneins), as well as SOD1, which serves as a reservoir of ligand-bound copper in the intracellular milieu as the excess copper may induce production of free radicals resulting in cellular destruction (3Thiele D.J. ACE1 regulates expression of the Saccharomyces cerevisiae metallothionein gene.Mol. Cell Biol. 1988; 8: 2745-2752Google Scholar). On the contrary, in the copper-deficit condition, the copper uptake genes are activated by Mac1 (4Pena M.M. Lee J. Thiele D.J. A delicate balance: homeostatic control of copper uptake and distribution.J. Nutr. 1999; 129: 1251-1260Google Scholar). Since the mammalian system inhabits a number of orthologs of copper metabolism–related enzymes in yeast, in-depth studies in this simple eukaryote have provided critical insights into the mammalian system. The mammalian orthologs of copper metabolism are stringently regulated by co-ordination of multiple transcription factors, which reflect maintenance of this essential metal in extremely different and unique levels from cells to organs (5Ehrensberger K.M. Bird A.J. Hammering out details: regulating metal levels in eukaryotes.Trends Biochem. Sci. 2011; 36: 524-531Google Scholar). As a result, any perturbation in copper metabolism results in serious consequences. For example, Menkes disease because of mutations in the ATP7A gene is characterized by intestinal copper accumulation, whereas its depletion in the enzymes of the peripheral region (6Nyasae L. Bustos R. Braiterman L. Eipper B. Hubbard A. Dynamics of endogenous ATP7A (menkes protein) in intestinal epithelial cells: copper-dependent redistribution between two intracellular sites.Am. J. Physiol. Gastrointest. Liver Physiol. 2007; 292: G1181-1194Google Scholar, 7Ravia J.J. Stephen R.M. Ghishan F.K. Collins J.F. Menkes Copper ATPase (Atp7a) is a novel metal-responsive gene in rat duodenum, and immunoreactive protein is present on brush-border and basolateral membrane domains.J. Biol. Chem. 2005; 280: 36221-36227Google Scholar) caused Wilson’s disease, because of mutations in the ATP7B gene, which is characterized by excessive copper accumulation in the neuronal and hepatic tissues (8Ralle M. Huster D. Vogt W. disease a intracellular copper in Biol. Chem. Scholar, a copper protein in disease and copper Cell. Scholar, R. The and disease proteins, have in and Biol. Chem. 2005; 280: Scholar). copper is through of uptake, and H. C. Copper metabolism in Saccharomyces an Scholar). Sec61 is a for protein by and post-translational the endoplasmic and 2007; Scholar). In yeast, the translocon is of proteins, and A.J. of the of yeast an essential of the endoplasmic Biol. Chem. Scholar, B. of a Scholar). The of the These are by out of which a in of proteins, whereas a which is and a R. of of in and Cell Biol. 2005; Scholar). the of is a protein of a region and a region to have factor is to a and its are yeast genes and that the of a be by expression of the of which the of a H. J. The of the endoplasmic translocon the in Saccharomyces Biol. Chem. Scholar, J. D. G. The of the of the endoplasmic as the factor for the of the Biol. Chem. Scholar, D. C. J. is for its the Sec61 and Biol. Chem. 2007; Scholar). is an essential protein of the which is an membrane protein its to the D. R. a of the Sec61 of the yeast protein Biol. Chem. Scholar). studies of the from different have revealed the through which the the to into the or the endoplasmic of from B. of a Scholar) and yeast Sec61 A. of yeast and mammalian Sec61 the Scholar) have revealed the in the from by to active by In its the by a and an of residues. the from the the bearing the the the in its are and the for the to The of a the to the entry of from the into the B. of a Scholar, A. of yeast and mammalian Sec61 the Scholar). In the of only the between Sec61 and the is required A. of yeast and mammalian Sec61 the Scholar, R. D. R. A. G. of for the in the Scholar). While for post-translational the The is of two essential and and two and R. of yeast proteins in into the endoplasmic into a Scholar, J. M. R. R. mutants in the of membrane proteins into the endoplasmic Biol. Cell. Scholar). of proteins, the secretory is in the in form. These proteins are translocated to are and post-translationally and then to further modifications to in the active M. M. M. D. B. The secretory yeast Scholar). For example, surface protein a is post-translationally through the of a This the of protein to the of the L. C. R. and of yeast in Biol. Chem. Scholar, J. L. of the Gas1 protein into the and is required for in Biol. Cell. Scholar). is in the of The in the through post-translational the is resulting in which the through and to the the is then to the are to the the to the mature CPY M. D. R. serves multiple in secretory and into the endoplasmic Biol. Cell. Scholar). the translocon may in the of the A number of from different have the of the of and post-translationally In of the been to the in in H. a inhibits the secretory by whereas of inhibits by the J. H. B. inhibition of of 2005; Scholar, A. protein by and Sec61 Biol. 2011; Scholar, A. J. Sec61 the Scholar). a from the been to the Sec61 translocon machinery of the yeast and The of and post-translationally Furthermore, mutations of the and of yeast and mammalian the these resistant mutants to the mutants of Sec61 and the of bearing secretory proteins, which are by the Sec61. that the binds to the translocon that to for the of proteins J. C. M. a protein the Cell Sci. Scholar). that copper the levels of secretory proteins in multiple of copper on cells: cellular and Scholar). The for the is While the of in or in the of in is its on the maturation of secretory proteins been M. in and 2011; Scholar, and Scholar, A. of and in protein Biol. Chem. Scholar). In an to the of these in the maturation of secretory proteins, we the of multiple on the secretory protein maturation and that copper to accumulation of form. In this we to the by the of copper on two different secretory proteins, Gas1 and We the accumulation of Gas1 by copper of yeast The accumulation of Gas1 protein by of cells and copper The of the protein revealed that the Gas1 protein as form. in yeast cells been to of proteins an to the endoplasmic Scholar). We that the of copper as the Furthermore, we that protein in the of copper, as a protein the the accumulation of M. R. of a secretory protein from the endoplasmic to the for J. Scholar, Collins D.J. R. The essential gene the of the yeast a protein in Cell Biol. Scholar). We copper to accumulation of protein in the then the be the and CPY utilize the Sec61 to gain into the further post-translational modifications J. L. of the Gas1 protein into the and is required for in Biol. Cell. Scholar, M. R. of a secretory protein from the endoplasmic to the for J. Scholar). 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Scholar). that the cells copper accumulation of Gas1 we to the of this form. that the cells in Gas1 the In the Gas1 is present the and the region in the cells only in the in the in the the of the protein is or the cells show an in the the we that the protein as the Gas1 protein copper of the revealed that is a As like Gas1 the to the endoplasmic Cell Biol. copper we that copper results in accumulation of copper to be in the cytosol. of cells copper cells an and then or of from these to in the as of which an of and then or of copper for endoplasmic surface on the we that the accumulation of in the of copper might be the of depletion binds to and in Chem. Scholar, R. M. R. G. of and of its in Saccharomyces Biol. Cell. Scholar) to a in the of to Gas1 R. M. R. G. of and of its in Saccharomyces Biol. Cell. 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Scholar) in copper and that one of the mutants is resistant to copper and This mutations in L80M, V134I, M248V, L342S and been to be in the of may that copper one or of these to its toxicity the the and the is resistant to the copper one of the the of two and which have been to as and mutants of the Sec61 protein have been to be resistant to the which the entry of or post-translationally into the These mutants the of the bearing of the the mutants to the conformation of the and the of cells resulting from of the of by the is ameliorated J. C. M. a protein the Cell Sci. Scholar). a number of mutants have been for the to the and region of the protein L. M. The of the Sec61 translocon the for membrane Biol. Cell. Scholar). If the the phenotype and is resistant to may be that copper binds to Sec61 is or before its that the is which inhibits the of the secretory test the of mutants in copper we the of which to be in of post-translationally proteins and phenotype J. C. 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In cells of Sec61 are to the such an inhibition the of Sec61 are in such the is ameliorated As Sec61 translocon of and of which the Sec61 and through its and to the of the translocon H. J. The of the endoplasmic translocon the in Saccharomyces Biol. Chem. Scholar). of the translocon be because of levels of Sec61 in be to out the of copper on or in the Copper been to a in the levels of the secretory proteins of copper on cells: cellular and Scholar). been for the a that the of the translocon the of these have that the protein in its in are in of secretory proteins and are to Furthermore, the of and CPY M. D. R. serves multiple in secretory and into the endoplasmic Biol. Cell. Scholar, G. M. A of factors proteins to the endoplasmic Scholar). We results the yeast cells Copper toxicity of Sec61 levels using the copper to of and CPY by affecting the through the Sec61 proteins are in many the cells as well as the proteins of many and of in 1988; Scholar, D.R. The of in J. Biochem. 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D. that regulates Scholar) few the yeast cells different conditions and the of cells in and of by to the The protein for The and the The in and for which for to the and the the protein for to the proteins to membrane in and for using from the membrane in for for The membrane in for which and to of the membrane then in for which to of the using system. for as from in from R. and The of Gas1 or CPY by the of mature and that of The to the Gas1 or CPY protein of protein protein as of of protein) for CPY the through of plasmid and the for CPY A. D.J. A. A. for and gene and in Saccharomyces Scholar). 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R. serves multiple in secretory and into the endoplasmic Biol. Cell. Scholar, J. C. M. a protein the Cell Sci. Scholar, M. R. of a secretory protein from the endoplasmic to the for J. Scholar, G. M. A of factors proteins to the endoplasmic Scholar, B. The Gas1 regulates in Saccharomyces Scholar, W. J. H. B. of enzymes and in Saccharomyces Biol. Cell. Scholar, Collins M. J. of the yeast Scholar). The that have of the of this We for SEC61 plasmid and its for the for for and for the We for in the of the and for This by from the of and of and and R. and R. R. R. and R. from

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