Advertisement

Association between the 15-kDa Selenoprotein and UDP-glucose:Glycoprotein Glucosyltransferase in the Endoplasmic Reticulum of Mammalian Cells*

Open AccessPublished:January 01, 2001DOI:https://doi.org/10.1074/jbc.M009861200
      Mammalian selenocysteine-containing proteins characterized with respect to function are involved in redox processes and exhibit distinct expression patterns and cellular locations. A recently identified 15-kDa selenoprotein (Sep15) has no homology to previously characterized proteins, and its function is not known. Here we report the intracellular localization and identification of a binding partner for this selenoprotein which implicate Sep15 in the regulation of protein folding. The native Sep15 isolated from rat prostate and mouse liver occurred in a complex with a 150-kDa protein. The latter protein was identified as UDP-glucose:glycoprotein glucosyltransferase (UGTR), the endoplasmic reticulum (ER)-resident protein, which was previously shown to be involved in the quality control of protein folding. UGTR functions by glucosylating misfolded proteins, retaining them in the ER until they are correctly folded or transferring them to degradation pathways. To determine the intracellular localization of Sep15, we expressed a green fluorescent protein-Sep15 fusion protein in CV-1 cells, and this protein was localized to the ER and possibly other perinuclear compartments. We determined that Sep15 contained the N-terminal signal peptide that was essential for translocation and that it was cleaved in the mature protein. However, C-terminal sequences of Sep15 were not involved in trafficking and retention of Sep15. The data suggest that the association between Sep15 and UGTR is responsible for maintaining the selenoprotein in the ER. This report provides the first example of the ER-resident selenoprotein and suggests a possible role of the trace element selenium in the quality control of protein folding.
      Sec
      selenocysteine
      Sep15
      15-kDa selenoprotein
      PAGE
      polyacrylamide gel electrophoresis
      UGTR
      UDP-glucose:glycoprotein glucosyltransferase
      ER
      endoplasmic reticulum
      HPLC
      high performance liquid chromatography
      GFP
      green fluorescent protein
      AEBSF
      4-(2-aminoethyl)benzenesulfonyl fluoride
      DTT
      dithiothreitol
      Selenium is an essential trace element in the diet of many organisms, including humans. It is present in the form of a selenocysteine (Sec)1 residue in several naturally occurring enzymes and proteins (
      • Low S.C.
      • Berry M.J.
      ,
      • Stadtman T.C.
      ). In selenoenzymes with established function, such as glutathione peroxidases, thyroid hormone deiodinases, and thioredoxin reductases in mammals, and hydrogenases and formate dehydrogenases in bacteria and archaea, Sec is present at the enzyme active center and participates in various redox reactions (
      • Stadtman T.C.
      ).
      Functions of many other mammalian selenoproteins, including the 15-kDa selenoprotein (Sep15), selenoprotein P, selenoprotein W, selenoprotein R (also named selenoprotein X), selenoprotein T, and selenoprotein N, have not been established. However, most of these proteins have clearly identifiable Sec-containing redox motifs, such as the Cys-Xaa-Xaa-Sec motif in selenoprotein W and selenoprotein T, suggesting their possible involvement in redox processes (
      • Gladyshev V.N.
      • Hatfield D.L.
      ).
      Sep15 was recently identified in human T-cells (
      • Gladyshev V.N.
      • Jeang K.T.
      • Wootton J.C.
      • Hatfield D.L.
      ). The gene for this protein is expressed in various human tissues with highest expression levels in the prostate and thyroid. In addition to humans, genes encoding Sep15 were detected in mice and rats. Sep15 exhibits no homology to previously characterized proteins, which precluded its functional characterization. However, Sep15 has a highly conserved motif, Cys-Gly-Sec-Lys, suggesting that this center could constitute an active center, in which Sec and Cys form a reversible seleno-sulfide bond. Besides this putative redox center, a previously noted feature in the Sep15 sequence was the lack of N-terminal sequences in the isolated human T-cell selenoprotein, which suggested the possibility of post-translational processing of the protein. In addition, Sep15 migrated as the 15-kDa protein on SDS-PAGE gels, whereas the migration properties of the native protein were consistent with a protein of ∼160–240 kDa. The low abundance of the 15-kDa selenoprotein in human T-cells and its lability during isolation did not permit isolation of the native protein to homogeneity to test whether the 160-kDa complex was composed of multiple selenoprotein subunits or if other protein components were involved in the complex (
      • Gladyshev V.N.
      • Jeang K.T.
      • Wootton J.C.
      • Hatfield D.L.
      ).
      The finding that the protein was expressed in the prostate at elevated levels compared with other tissues (
      • Gladyshev V.N.
      • Jeang K.T.
      • Wootton J.C.
      • Hatfield D.L.
      ,
      • Kumaraswamy E.
      • Malykh A.
      • Korotkov K.V.
      • Kozyavkin S.
      • Hu Y.
      • Kwon S.Y.
      • Moustafa M.E.
      • Carlson B.A.
      • Berry M.J.
      • Lee B.J.
      • Hatfield D.L.
      • Diamond A.M.
      • Gladyshev V.N.
      ) provided an opportunity to determine the oligomeric composition of Sep15 by isolating the selenoprotein from this organ. In this report, we describe isolation of Sep15 from rat prostate and mouse liver. In both preparations, the native selenoprotein occurred as a complex with UDP-glucose:glycoprotein glucosyltransferase (UGTR), an enzyme involved in the quality control of protein folding (
      • Parodi A.J.
      ). Further characterization revealed that Sep15 was located in perinuclear cellular compartments, consistent with the finding that UGTR is located in the endoplasmic reticulum (ER). The observation that Sep15 was found only in a complex with UGTR suggests that it may be involved in the regulation of protein folding.

      DISCUSSION

      In this report, we described the association between Sep15 and UGTR in the ER of mammalian cells. The data show that Sep15 is tightly bound to UGTR, which suggests that this selenoprotein may be linked to the quality control of protein folding.
      Sep15 had previously been isolated only from a human T-cell line and only under denaturing conditions (
      • Gladyshev V.N.
      • Jeang K.T.
      • Wootton J.C.
      • Hatfield D.L.
      ). Thus, a possibility remained that Sep15 was a component of a multiprotein complex or a homomultimer. Isolation of Sep15 from mammalian tissues and cell lines was proven to be difficult because of its extreme lability and low abundance. However, taking advantage of the finding that Sep15 exhibits high expression levels in prostate (
      • Gladyshev V.N.
      • Jeang K.T.
      • Wootton J.C.
      • Hatfield D.L.
      ), we isolated the protein from rat prostate. A procedure for isolation of Sep15 was developed which combined conventional chromatography and HPLC. This procedure allowed rapid isolation of the protein and minimized losses through denaturation.
      The isolated selenoprotein was found to occur in a complex with a 150-kDa protein. Comparison of sequenced peptides from the 150-kDa protein with the deduced sequence of rat liver UGTR revealed 100% identity in four peptide sequences. Western blot analyses of the 150-kDa protein with the anti-UGTR antibodies, as well as analyses of fractionated mouse liver extracts, further supported the conclusion that Sep15 was purified in a complex with UGTR. This finding was unexpected for the following reasons. (i) UGTR is known for its role in the quality control of protein folding (
      • Parodi A.J.
      ). This enzyme recognizes misfolded protein domains in the ER lumen of eukaryotic cells and specifically glucosylates these proteins, which retains misfolded proteins in the ER for the next cycle of folding by the calnexin/calreticulin glycoprotein folding system (
      • Trombetta E.S.
      • Helenius A.
      ,
      • Ritter C.
      • Helenius A.
      ,
      • Matouschek A.
      ). Previously characterized eukaryotic selenoproteins were involved in redox processes (
      • Stadtman T.C.
      ), and redox function has been anticipated for Sep15, but the quality control of protein folding has not been linked to a redox process. (ii) UGTR is located in the ER (
      • Trombetta S.E.
      • Ganan S.A.
      • Parodi A.J.
      ), and no selenoprotein has yet been found to occur in this cellular compartment. In addition to Sep15, two other known mammalian selenoproteins, glutathione peroxidase 3 and selenoprotein P, contain N-terminal signal peptides. These proteins are secreted and are the major selenoproteins in the plasma of mammals (
      • Burk R.F.
      • Hill K.E.
      ). Most other known mammalian selenoproteins are cytosolic, nuclear, or mitochondrial proteins.
      To determine the intracellular localization of Sep15, we made a series of constructs that encoded fusion proteins between Sep15 and GFP. In addition, we tested the relevance of the N-terminal portion of Sep15 for the ER translocation and of the C-terminal portion of the protein for ER retention. Expression patterns of the GFP fusion constructs containing Sep15 sequences in CV-1 cells were examined and compared with location of a marker by live-cell imaging confocal microscopy. Although the marker that was used in the present study is known to label both ER and Golgi, the fact that UGTR is the ER resident protein strongly suggests that Sep15 colocalized with UGTR in the ER. The data from imaging analyses in combination with other biochemical evidence demonstrated that the N-terminal signal peptide of Sep15 was necessary for ER localization. In contrast, the C-terminal tetrapeptide of the selenoprotein lacked a typical ER retention signal, and this sequence was not necessary to keep the protein in the ER. It appears that the selenoprotein sequence itself was responsible for retaining Sep15 in the ER and preventing its secretion. The data thus suggested that Sep15 was maintained in the ER because of its interaction with UGTR.
      The binding between Sep15 and UGTR appeared to be very strong because these proteins copurified at each isolation step. Moreover, Sep15 was found exclusively in the UGTR-bound form. The lack of the UGTR-free selenoprotein may also be consistent with the idea that Sep15 and UGTR are subunits of a two-subunit protein. It is possible that the presence of the selenoprotein subunit in UGTR preparations was unnoticed previously because of the small size of Sep15, which made it difficult to visualize the selenoprotein by protein staining on SDS-PAGE gels. In addition, low percentage SDS-PAGE gels have been used previously for homogeneity assessment of isolated UGTR (
      • Trombetta S.E.
      • Parodi A.J.
      ). In these gels, selenoprotein would migrate in the dye front.
      In contrast to the exclusive binding of Sep15 to UGTR, the latter protein was detected in both selenoprotein-bound and selenoprotein-free forms. It remains to be determined if the selenoprotein-free form arose by the release of the denatured selenoprotein during protein isolation, if it was a natural UGTR form or if UGTR also occurred in a complex with other proteins and/or selenoproteins.
      UGTR was shown previously, by immunoprecipitation, to associate with misfolded proteins, such as α1-antitrypsin (
      • Choudhury P.
      • Liu Y.
      • Bick R.J.
      • Sifers R.N.
      ), and with other ER resident proteins, such as protein disulfide isomerases, carboxylesterase, and the glucose-regulated protein (
      • Amouzadeh H.R.
      • Bourdi M.
      • Martin J.L.
      • Martin B.M.
      • Pohl L.R.
      ). However, these proteins do not copurify with UGTR, and only a small fraction of them was associated with this enzyme. Sep15, on the other hand, was found exclusively in the UGTR-bound form in rat prostate and mouse liver.
      UGTR is the only known quality control protein that recognizes misfolded proteins in the ER, and its mechanism has been characterized in great detail. Interestingly, UGTR is able to glucosylate misfolded domains specifically while not reacting with properly folded domains within a protein composed of identical folded and misfolded domains (
      • Ritter C.
      • Helenius A.
      ).
      The possible role of redox processes in the ER-based protein folding has received much attention recently. In particular, protein disulfide isomerase was found to remove electrons, through the disulfide bond formation, from folding proteins and to transfer reducing equivalents further to the ER membrane protein Ero1 (
      • Pollard M.G.
      • Travers K.J.
      • Weissman J.S.
      ,
      • Frand A.R.
      • Cuozzo J.W.
      • Kaiser C.A.
      ). The formation of disulfide bonds in nascent polypeptides is believed to be associated with folding by the calnexin/calreticulin chaperones. Although properly folded proteins may proceed further to secretory pathways, misfolded polypeptides including those containing disulfide bonds are glucosylated by UGTR to retain them for the next cycle of folding. Sensing or reduction of disulfides within misfolded proteins prior to folding appears to be required. Whether Sep15 is involved in such redox reactions is a direction for further research.

      Acknowledgments

      We thank Dr. Armando Parodi (Instituto de Investigaciones Biotecnologicas, Universidad de San Martin, Buenos Aires, Argentina) for providing anti-UGTR antibodies and an unpublished sequence of rat liver UGTR.

      REFERENCES

        • Low S.C.
        • Berry M.J.
        Trends Biochem. Sci. 1996; 21: 203-208
        • Stadtman T.C.
        Annu. Rev. Biochem. 1996; 65: 83-100
        • Gladyshev V.N.
        • Hatfield D.L.
        J. Biomed. Sci. 1999; 6: 151-160
        • Gladyshev V.N.
        • Jeang K.T.
        • Wootton J.C.
        • Hatfield D.L.
        J. Biol. Chem. 1998; 273: 8910-8915
        • Kumaraswamy E.
        • Malykh A.
        • Korotkov K.V.
        • Kozyavkin S.
        • Hu Y.
        • Kwon S.Y.
        • Moustafa M.E.
        • Carlson B.A.
        • Berry M.J.
        • Lee B.J.
        • Hatfield D.L.
        • Diamond A.M.
        • Gladyshev V.N.
        J. Biol. Chem. 2000; 275: 35540-35547
        • Parodi A.J.
        Biochem. J. 2000; 348: 1-13
        • Gladyshev V.N.
        • Factor V.M.
        • Housseau F.
        • Hatfield D.L.
        Biochem. Biophys. Res. Commun. 1998; 251: 488-493
        • Trombetta S.E.
        • Parodi A.J.
        J. Biol. Chem. 1992; 267: 9236-9240
        • Kryukov G.V.
        • Kryukov V.M.
        • Gladyshev V.N.
        J. Biol. Chem. 1999; 274: 33888-33897
        • Kok L.W.
        • Babia T.
        • Klappe K.
        • Egea G.
        • Hoekstra D.
        Biochem. J. 1998; 333: 779-786
        • Ilgoutz S.C.
        • Mullin K.A.
        • Southwell B.R.
        • McConville M.J.
        EMBO J. 1999; 18: 3643-3654
        • Parker C.G.
        • Fessler L.I.
        • Nelson R.E.
        • Fessler J.H.
        EMBO J. 1995; 14: 1294-1303
        • Tessier D.
        • Dignard D.
        • Zapun A.
        • Radominska-Pandya A.
        • Parodi A.J.
        • Bergeron J.J.
        • Thomas D.Y.
        Glycobiology. 2000; 10: 403-412
        • Arnold S.M.
        • Fessler L.I.
        • Fessler J.H.
        • Kaufman R.J.
        Biochemistry. 2000; 39: 2149-2163
        • Trombetta E.S.
        • Helenius A.
        Curr. Opin. Struct. Biol. 1998; 8: 587-592
        • Ritter C.
        • Helenius A.
        Nat. Struct. Biol. 2000; 7: 278-280
        • Trombetta S.E.
        • Ganan S.A.
        • Parodi A.J.
        Glycobiology. 1991; 1: 155-161
        • Berry M.J.
        • Banu L.
        • Harney J.W.
        • Larsen P.R.
        EMBO J. 1993; 12: 3315-3322
        • Tujebajeva R.M.
        • Harney J.W.
        • Berry M.J.
        J. Biol. Chem. 2000; 275: 6288-6294
        • Fukasawa M.
        • Nishijima M.
        • Hanada K.
        J. Cell Biol. 1999; 144: 673-685
        • Matouschek A.
        Nat. Struct. Biol. 2000; 7: 265-266
        • Burk R.F.
        • Hill K.E.
        Bioessays. 1999; 21: 231-237
        • Choudhury P.
        • Liu Y.
        • Bick R.J.
        • Sifers R.N.
        J. Biol. Chem. 1997; 272: 13446-13451
        • Amouzadeh H.R.
        • Bourdi M.
        • Martin J.L.
        • Martin B.M.
        • Pohl L.R.
        Chem. Res. Toxicol. 1997; 10: 59-63
        • Pollard M.G.
        • Travers K.J.
        • Weissman J.S.
        Mol. Cell. 1998; 1: 171-182
        • Frand A.R.
        • Cuozzo J.W.
        • Kaiser C.A.
        Trends Cell Biol. 2000; 10: 203-210