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• W4283216927 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 S. Schirrmeister W. Burkhead J.L. Capps T.R. et al.Wilson disease at a single cell level: intracellular copper trafficking activates compartment-specific responses in hepatocytes.J. Biol. Chem. 2010; 285: 30875-30883Google Scholar, 9Mufti A.R. Burstein E. Csomos R.A. Graf P.C. Wilkinson J.C. Dick R.D. et al.XIAP Is a copper binding protein deregulated in Wilson's disease and other copper toxicosis disorders.Mol. Cell. 2006; 21: 775-785Google Scholar, 10Barnes N. Tsivkovskii R. Tsivkovskaia N. Lutsenko S. The copper-transporting ATPases, menkes and wilson disease proteins, have distinct roles in adult and developing cerebellum.J. Biol. Chem. 2005; 280: 9640-9645Google Scholar). Thus, copper homeostasis is maintained through tight coordination of uptake, transport, and excretion (11Shi H. Jiang Y. Yang Y. Peng Y. Li C. Copper metabolism in Saccharomyces cerevisiae: an update.Biometals. 2021; 34: 3-14Google Scholar). Sec61 complex is a heterotrimeric channel for protein conduction used by both cotranslational and post-translational translocation pathways (12Rapoport T.A. Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes.Nature. 2007; 450: 663-669Google Scholar). In yeast, the translocon is composed of three proteins, Sbh1, Sec61, and Sss1 (13Wilkinson B.M. Critchley A.J. Stirling C.J. Determination of the transmembrane topology of yeast Sec61p, an essential component of the endoplasmic reticulum translocation complex.J. Biol. Chem. 1996; 271: 25590-25597Google Scholar, 14Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. et al.X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Google Scholar). The core structure of the Sec61p, approximately 53 kDa protein, comprises 10 transmembrane (TM) helices. These helices are separated by loops, out of which L6 plays a role in translocation of proteins, whereas L8 contains a highly conserved residue, which is positively charged and provides a major ribosome-binding site (15Cheng Z. Jiang Y. Mandon E.C. Gilmore R. Identification of cytoplasmic residues of Sec61p involved in ribosome binding and cotranslational translocation.J. Cell Biol. 2005; 168: 67-77Google Scholar). Sbh1, the beta subunit of translocon, is a small protein of approximately 8.7 kDa. Structurally, it has a single TM domain at C-terminal region and a cytosolic N-terminal region previously thought to have guanine nucleotide exchange factor (GEF) activity. However, it is no longer believed to contain a GEF activity. Both Sbh1 and its homolog Sbh2 are nonessential yeast genes and that the translocation defects of a Δsbh1Δsbh2 mutant can be suppressed by expression of the TM span of Sbh1, which argues against the presence of a cytoplasmic GEF domain (16Toikkanen J.H. Miller K.J. Soderlund H. Jantti J. Keranen S. The beta subunit of the Sec61p endoplasmic reticulum translocon interacts with the exocyst complex in Saccharomyces cerevisiae.J. Biol. Chem. 2003; 278: 20946-20953Google Scholar, 17Helmers J. Schmidt D. Glavy J.S. Blobel G. Schwartz T. The beta-subunit of the protein-conducting channel of the endoplasmic reticulum functions as the guanine nucleotide exchange factor for the beta-subunit of the signal recognition particle receptor.J. Biol. Chem. 2003; 278: 23686-23690Google Scholar, 18Feng D. Zhao X. Soromani C. Toikkanen J. Romisch K. Vembar S.S. et al.The transmembrane domain is sufficient for Sbh1p function, its association with the Sec61 complex, and interaction with Rtn1p.J. Biol. Chem. 2007; 282: 30618-30628Google Scholar). Sss1 is an essential small protein of the complex, which is an integral membrane protein with its amino terminal exposed to the cytosol (19Esnault Y. Feldheim D. Blondel M.O. Schekman R. Kepes F. SSS1 encodes a stabilizing component of the Sec61 subcomplex of the yeast protein translocation apparatus.J. Biol. Chem. 1994; 269: 27478-27485Google Scholar). Multiple structural studies of the trimeric complex from different species have revealed details about the mechanism through which the polypeptides enter the protein-conducting channel to reach into the lumen or become associated with the endoplasmic reticulum (ER) membrane. X-ray structures of protein-conducting channel from Methanococcus jannaschii (14Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. et al.X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Google Scholar) and monomeric yeast Sec61 complexes (20Becker T. Bhushan S. Jarasch A. Armache J.P. Funes S. Jossinet F. et al.Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.Science. 2009; 326: 1369-1373Google Scholar) have revealed the plausible structural changes in the translocation when shifting from idle (not engaged by polypeptide) state to active state (engaged by polypeptide). In its idle state, the channel pore remains closed by a plug domain and an additional ring of hydrophobic residues. Together, they block the outer cytoplasmic environment from the inner ER lumen. However, when the signal sequence bearing polypeptide approaches the channel, the interactions holding the plug in its position are destabilized, and so the plug shifts making room for the polypeptide to pass through. The ring of hydrophobic residues forms a seal around the passing polypeptide to prevent the entry of small molecules from the cytoplasm into the lumen (14Van den Berg B. Clemons Jr., W.M. Collinson I. Modis Y. Hartmann E. Harrison S.C. et al.X-ray structure of a protein-conducting channel.Nature. 2004; 427: 36-44Google Scholar, 20Becker T. Bhushan S. Jarasch A. Armache J.P. Funes S. Jossinet F. et al.Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.Science. 2009; 326: 1369-1373Google Scholar). In the case of cotranslational translocation, only the association between protein-conducting Sec61 and the ribosome’s exit tunnel is required (20Becker T. Bhushan S. Jarasch A. Armache J.P. Funes S. Jossinet F. et al.Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.Science. 2009; 326: 1369-1373Google Scholar, 21Beckmann R. Bubeck D. Grassucci R. Penczek P. Verschoor A. Blobel G. et al.Alignment of conduits for the nascent polypeptide chain in the ribosome-Sec61 complex.Science. 1997; 278: 2123-2126Google Scholar). While for post-translational translocation, the complex partners with Sec62–Sec63 complex. The Sec62–Sec63 complex is composed of two essential subunits, Sec62p and Sec63p, and two nonessential subunits, Sec71p and Sec72p (22Deshaies R.J. Sanders S.L. Feldheim D.A. Schekman R. Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex.Nature. 1991; 349: 806-808Google Scholar, 23Stirling C.J. Rothblatt J. Hosobuchi M. Deshaies R. Schekman R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.Mol. Biol. Cell. 1992; 3: 129-142Google Scholar). Plethora of proteins, utilizing the secretory pathway, is synthesized in the cytosol in their inactive form. These proteins are translocated to ER, where they are folded and post-translationally modified and then transferred to Golgi, where further modifications take place to finally reach their diverse destinations in the active form (24Delic M. Valli M. Graf A.B. Pfeffer M. Mattanovich D. Gasser B. The secretory pathway: exploring yeast diversity.FEMS Microbiol. Rev. 2013; 37: 872-914Google Scholar). For example, glycophospholipid-anchored surface protein (Gas1), a β-(1, 3)-glucan elongase protein, is post-translationally modified through the attachment of a glycolipid moiety called glycosylphosphatidylinositol (GPI) at their C-terminal end. This attachment facilitates the anchoring of protein to the plasma membrane’s outer leaflet of the lipid bilayer (25Popolo L. Ragni E. Carotti C. Palomares O. Aardema R. Back J.W. et al.Disulfide bond structure and domain organization of yeast beta(1,3)-glucanosyltransferases involved in cell wall biogenesis.J. Biol. Chem. 2008; 283: 18553-18565Google Scholar, 26Rolli E. Ragni E. Calderon J. Porello S. Fascio U. Popolo L. Immobilization of the glycosylphosphatidylinositol-anchored Gas1 protein into the chitin ring and septum is required for proper morphogenesis in yeast.Mol. Biol. Cell. 2009; 20: 4856-4870Google Scholar). Similarly, carboxypeptidase Y (CPY) is synthesized in the form of preproenzyme. The preproenzyme enters in the ER lumen through post-translational translocation, where the signal sequence is cleaved resulting in proCPY after which the enzyme gets folded through disulphide bond formation and glycosylation to give the p1CPY form; the enzyme is then transported to Golgi, where the outer mannose residues are added giving rise to p2CPY. Finally, inside the vacuole, proteinase B cleaves the p2CPY to form the mature CPY (27Pilon M. Romisch K. Quach D. Schekman R. Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane.Mol. Biol. Cell. 1998; 9: 3455-3473Google Scholar). Molecules specifically inhibiting the translocon channel may aid in elucidating the underlying functional mechanisms of the complex. A number of molecules from different sources have shown the potential of inhibiting the translocation of cotranslationally and post-translationally targeting proteins. In case of apratoxin A, the molecule has been shown to hamper the cotranslational translocation in in vitro condition (28Liu Y. Law B.K. Luesch H. Apratoxin a reversibly inhibits the secretory pathway by preventing cotranslational translocation.Mol. Pharmacol. 2009; 76: 91-104Google Scholar), whereas group of molecules called cotransins inhibits specific translocating substrates by targeting the Sec61α (29Besemer J. Harant H. Wang S. Oberhauser B. Marquardt K. Foster C.A. et al.Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1.Nature. 2005; 436: 290-293Google Scholar, 30Maifeld S.V. MacKinnon A.L. Garrison J.L. Sharma A. Kunkel E.J. Hegde R.S. et al.Secretory protein profiling reveals TNF-alpha inactivation by selective and promiscuous Sec61 modulators.Chem. Biol. 2011; 18: 1082-1088Google Scholar, 31Mackinnon A.L. Paavilainen V.O. Sharma A. Hegde R.S. Taunton J. An allosteric Sec61 inhibitor traps nascent transmembrane helices at the lateral gate.Elife. 2014; 3e01483Google Scholar). More recently, a new natural metabolite isolated from the organism Chaetosphaeria tulasneorum has been shown to target the Sec61 translocon machinery of the yeast and mammals. The translocation of both cotranslationally and post-translationally translocating substrates was inhibited. Furthermore, dominant mutations of Sec61, isolated, post the mutagenesis and genome sequencing of yeast and mammalian cells, conferred resistance against the isolated metabolite. Finally, these resistant mutants were shown to exhibit the prl phenotype. prl mutants mimic partially opened channel of Sec61 and so allow the passing of defective signal bearing secretory proteins, which are otherwise blocked by the WT Sec61. Thus, it was concluded that the metabolite most probably binds to the closed translocon structure so that it fails to open for the efficient translocation of proteins (32Junne T. Wong J. Studer C. Aust T. Bauer B.W. Beibel M. et al.Decatransin, a new natural product inhibiting protein translocation at the Sec61/SecYEG translocon.J. Cell Sci. 2015; 128: 1217-1229Google Scholar). Reports suggest that copper effects the glycosylation levels of secretory proteins in multiple cell lines (33Yuk I.H. Russell S. Tang Y. Hsu W.T. Mauger J.B. Aulakh R.P. et al.Effects of copper on CHO cells: cellular requirements and product quality considerations.Biotechnol. Prog. 2015; 31: 226-238Google Scholar). The underlying reason for the phenomenon is still obscure. While the role of transition metals in reactive oxygen species (ROS) generation or in the replacement of metals in metallothioneins is extensively studied, its effect on the maturation of secretory proteins has never been explored (34Jomova K. Valko M. Advances in metal-induced oxidative stress and human disease.Toxicology. 2011; 283: 65-87Google Scholar, 35Gaetke L.M. Chow-Johnson H.S. Chow C.K. Copper: Toxicological relevance and mechanisms.Arch. Toxicol. 2014; 88: 1929-1938Google Scholar, 36Meucci E. Mordente A. Martorana G.E. Metal-catalyzed oxidation of human serum albumin: conformational and functional changes. Implications in protein aging.J. Biol. Chem. 1991; 266: 4692-4699Google Scholar). In an attempt to explore the possible role of these metals in the maturation of secretory proteins, we tested the effect of multiple metals on the secretory protein maturation (Gas1, data not shown) and found that treatment with copper leads to accumulation of immature form. In this communication, we attempt to address the question by checking the effect of copper on two different secretory proteins, Gas1 and CPY. We first report the selective accumulation of immature Gas1 by copper (CuCl2.2H2O) treatment of yeast cells. The accumulation of immature Gas1 protein was reversed by treatment of cells with ethanolamine (ETA) and specific copper chelator bathocuproinedisulfonic acid disodium salt (BCS). The molecular weight of the immature protein revealed that the Gas1 protein was throttled as unglycosylated form. Tunicamycin (Tm) treatment in WT yeast cells has been shown to accumulate unglycosylated form of red fluorescent protein-Gas1 (37Aviram N. Ast T. Costa E.A. Arakel E.C. Chuartzman S.G. Jan C.H. et al.The SND proteins constitute an alternative targeting route to the endoplasmic reticulum.Nature. 2016; 540: 134-138Google Scholar). We found that the form of copper accumulated showed similar migration as the Tm-treated Gas1. Furthermore, we found that another signal recognition particle–independent protein CPY, in the presence of copper, accumulated as a slower migrating protein band than the Tm-treated bands pointing at the accumulation of proproteins (38Pilon M. Schekman R. Romisch K. Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.EMBO J. 1997; 16: 4540-4548Google Scholar, 39Silberstein S. Collins P.G. Kelleher D.J. Gilmore R. The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms.J. Cell Biol. 1995; 131: 371-383Google Scholar). We reasoned, if copper treatment leads to accumulation of protein in the prepro form, then the compound must be targeting the translocon. Both Gas1p and CPY utilize the Sec61 complex to gain access into the ER lumen where further post-translational modifications occur (26Rolli E. Ragni E. Calderon J. Porello S. Fascio U. Popolo L. Immobilization of the glycosylphosphatidylinositol-anchored Gas1 protein into the chitin ring and septum is required for proper morphogenesis in yeast.Mol. Biol. Cell. 2009; 20: 4856-4870Google Scholar, 38Pilon M. Schekman R. Romisch K. Sec61p mediates export of a misfolded secretory protein from the endoplasmic reticulum to the cytosol for degradation.EMBO J. 1997; 16: 4540-4548Google Scholar). On testing various components of the translocon complex, cells with lower levels of Sec61p showed compromised growth in the presence of copper. The growth-sensitive phenotype of Sec61 mutant in copper is rescued upon restoration of Sec61 levels through plasmid. The sec61-DAmP yeast cells reported to accumulate precursor Gas1p (40Ast T. Cohen G. Schuldiner M. A network of cytosolic factors targets SRP-independent proteins to the endoplasmic reticulum.Cell. 2013; 152: 1134-1145Google Scholar) also accumulated the preproCPY. The immature protein band of CPY corresponds to the band accumulated under copper treatment. We also tested Sec61 translocation-defective mutants (27Pilon M. Romisch K. Quach D. Schekman R. Sec61p serves multiple roles in secretory precursor binding and translocation into the endoplasmic reticulum membrane.Mol. Biol. Cell. 1998; 9: 3455-3473Google Scholar) in copper and found that only sec61-22, inhabiting mutations at L80M, V134I, M248V, and L342S, was resistant to copper. Thus, our results strongly suggest that copper may inhibit the Sec61 translocon–mediated entry/release of these proteins into the ER lumen. To investigate the effect of copper on Gas1p maturation, BY4743 yeast cells transformed with pRS415 Gas1-GFP (41Ha C.W. Kim K. Chang Y.J. Kim B. Huh W.K. The beta-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.Nucl. Acids Res. 2014; 42: 8486-8499Google Scholar) were treated with different copper concentrations, viz. 0.25, 0.5, and 1 mM, and cells were harvested at different time points, viz. 30′, 60′, and 90’. Following the harvest, we extracted the proteins and performed immunoblotting. Our result (Fig. 1A) clearly shows that the accumulation of precursor Gas1p is proportional to the concentration of copper used, and the time for which cells were exposed to copper (Fig. 1, A and D) shows that treatment with 1 mM copper at 90′ led to accumulation of the highest amount of Gas1p precursor form at 58% ± 14% compared with the untreated (UT) at 31% ± 13%. BCS is a specific copper chelator that is routinely used in yeast biology to reverse the effect of copper (42Dong K. Addinall S.G. Lydall D. Rutherford J.C. The yeast copper response is regulated by DNA damage.Mol. Cell Biol. 2013; 33: 4041-4050Google Scholar). To prove that copper is responsible for the accumulation of the immature protein, we cotreated cells with copper and BCS. The result (Fig. 1B) shows that indeed the addition of BCS rescues the accumulation of precursor form. While UT or copper-treated cells accumulated immature proteins at 24% ± 5% and 58% ± 2%, respectively; addition of BCS reduced the accumulation to about 32% ± 14% or 34% ± 14% at concentrations of 1 and 1.5 mM BCS, respectively (Fig. 1E). Furthermore, we checked another copper chelator, ETA, which is known to form complex with copper (43Caumul P. Boodhoo K. Burkutally S.B. Seeruttun S. Namooya N. Ramsahye N. et al.Synthesis and analysis of metal chelating amino and diamine precursors and their complex formation on copper (II) using conductivity and spectroscopic methods.Res. J. Pharm. Biol. Chem. Sci. 2014; 5: 494-509Google Scholar). Upon cosupplementation, the accumulation of immature form was reversed in a dose-dependent manner (Fig. 1, C and F). We found while the UT and copper-treated cells accumulate about 20% ± 6% and 61% ± 7% immature form, respectively. Cotreatment with ETA at 2.5 mM concentration reduced the accumulation to 45% ± 7%. Spot test assay of untransformed BY4743 cells showed that the deleterious effect on cell growth by copper was ameliorated by BCS (Fig. 1G). We did growth curve analysis of untransformed BY4743 cells and found that 1 mM copper was able to exert effect on the growth of cells in liquid culture, which was ameliorated by cotreatment of ETA and BCS, respectively (Fig. 1, H and I). We also checked the response of the transformed cells in the presence of copper, which inhibited the growth with increasing concentration (Fig. S1A). Since Gas1p was tagged with GFP, we next tested the localization of Gas1-GFP signal in the cell through microscopy. To this end, we transformed the GSHY583 strain containing the N-terminal ER signal sequence, dsRed, and an HDEL tag (44Suresh H.G. da Silveira Dos Santos A.X. Kukulski W. Tyedmers J. Riezman H. Bukau B. et al.Prolonged starvation drives reversible sequestration of lipid biosynthetic enzymes and organelle reorganization in Saccharomyces cerevisiae.Mol. Biol. Cell. 2015; 26: 1601-1615Google Scholar). After confirming that the transformed GSHY583 cells treated with copper showed accumulation of Gas1 precursor form (Fig. 2A), we performed microscopy to assess the localization of this precursor form. Figure 2B shows that the copper-treated cells exhibited marked difference in Gas1 distribution compared with the UT cells. In the UT cells, Gas1 is present at the nuclear rim and the cortical ER region in the mother cells but only in the cortical ER in the new buds. However, in the copper-treated cells, the cortical distribution of the protein is majorily distorted with almost no puncta or tubes. Moreover, the copper-treated cells show an increase in the diffused cytoplasmic Gas1-GFP signal compared with the UT cells. Interestingly, we found that the signal sequence–attached DsRed protein also exhibited similar pattern as the Gas1 protein (Fig. 2B) under copper treatment. Inspection of the signal sequence–attached dsRed-HDEL revealed that it is a Kar2 signal sequence. As Kar2 partly utilizes signal recognition particle-independent translocation like Gas1 (45Ng D.T. Brown J.D. Walter P. Signal sequences specify the targeting route to the endoplasmic reticulum membrane.J. Cell Biol. 1996; 134: 269-278Google Scholar), it shows defective distribution upon copper treatment. Thus, we conclude that copper treatment results in accumulation of immature Gas1p.Figure 2Immature Gas1p accumulated under copper appears to be in the cytosol. A, immunoblots of Gas1-GFP–transformed GSHY583 cells treated with copper CuCl2.2H2O (1 mM). Transformed cells were grown till an absorbance reached ∼1 at 600 nm and then left untreated (−) or treated with indicated concentration of copper. Samples were collected at 90’. Proteins extracted from these samples were subjected to Western blotting (described in the Experimental procedures section). TBP served as control. B, microscopy images of Gas1-GFP–transformed GSHY583 cells, which were grown till an absorbance of ∼1 at 600 nm and then left untreated (UT) or treated with indicated concentration of copper for 90’. Bar represents 5 μm. ER, endoplasmic reticulum; Gas1, glycophospholipid-anchored surface protein; TBP, TATA-binding protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on the existing studies, we hypothesized that the accumulation of immature Gas1p in the presence of copper might be the result of phosphatidylethanolamine pool depletion (46Poyton M.F. Sendecki A.M. Cong X. Cremer P.S. Cu(2+) binds to phosphatidylethanolamine and increases oxidation in lipid membranes.J. Am. Chem. Soc. 2016; 138: 1584-1590Google Scholar, 47Birner R. Burgermeister M. Schneiter R. Daum G. Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.Mol. Biol. Cell. 2001; 12: 997-1007Google Scholar) leading to a compromise in the addition of GPI anchor to Gas1 (47Birner R. Burgermeister M. Schneiter R. Daum G. Roles of phosphatidylethanolamine and of its several biosynthetic pathways in Saccharomyces cerevisiae.Mol. Biol. Cell. 2001; 12: 997-1007Google Scholar). To test this hypothesis, we checked the maturation of CPY, a non-GPI–anchored secretory protein. To this end, we endogenously myc tagged the CPY protein in BY4741 and tested the effect of copper on its growth (Fig. S1B). We then checked the effect of copper on CPY protein maturation. Results (Fig. 3, A and E) show the accumulation of immature CPY protein band under copper treatment, thereby ruling out the possibility that the absence of GPI anchor attachment might be causing the accumulation of immature Gas1p. The UT cells accumulated 15% ± 1%, whereas copper-treated cells accumulated 60% ± 8% and 66% ± 8% immature protein at concentrations of 0.75 and 1 mM, respectively. Slowly migrating bands of both, Gas1p and CPY, resembled the size of unglycosylated forms of these proteins. We debated, if the faster migrating band obtained under copper treatment is indeed the unglycosylated form, then" @default.
• W4283216927 created "2022-06-22" @default.
• W4283216927 creator A5007529008 @default.
• W4283216927 creator A5017878103 @default.
• W4283216927 date "2022-08-01" @default.
• W4283216927 modified "2023-10-01" @default.
• W4283216927 title "Copper inhibits protein maturation in the secretory pathway by targeting the Sec61 translocon in Saccharomyces cerevisiae" @default.
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• W4283216927 doi "https://doi.org/10.1016/j.jbc.2022.102170" @default.
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