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J Biol Chem. 1991 Oct 15;266(29):19599-610.

SSR alpha and associated calnexin are major calcium binding proteins of the endoplasmic reticulum membrane.

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  • 1Department of Anatomy, McGill University, Montreal, Quebec, Canada.

Abstract

GTP phosphorylation of rough microsomes in vitro is limited to four integral membrane proteins. Two of these, a phosphoprotein (pp90) and a phosphoglycoprotein (pgp35) were purified as a complex with two nonphosphorylated membrane glycoproteins, gp25H and gp25L. The authenticity of this complex was confirmed using two different purification procedures and by coimmunoprecipitation. By immunofluorescence a reticulated cytoplasmic network was revealed for the proteins which was similar to that for Louvard et al. (Louvard, D., Reggio, H., and Warren, G. (1982) J. Cell Biol. 92, 92-107) marker antisera which also recognized purified pp90 on immunoblots. Amino acid sequencing of peptides derived from pgp35 identified this protein as SSR alpha, an endoplasmic reticulum constituent as identified by cross-linking of translocating nascent chains (Görlich, D, Prehn, S., Hartmann, E., Herz, J., Otto, A., Kraft, R., Wiedmann, M., Knespel, S., Dobberstein, B., and Rapoport, T. A. (1990) J. Cell Biol. 111, 2283-2294). The sequence of gp25H was determined from cDNA clones and was identical with SSR beta identified by Görlich et al. (1990) as being tightly bound to SSR alpha. Sequencing of gp25L revealed no similarity of the deduced sequence with other proteins. However, pp90 revealed a high degree of sequence identity with the Ca(2+)-binding protein, calreticulin. 45Ca2+ overlay studies indicated that pp90 bound Ca2+ and the name calnexin is proposed. Surprisingly, pgp25 (SSR alpha) also bound Ca2+ although gp25H (SSR beta) and gp25L did not. Triton X-114 partitioning of the integral membrane proteins of rough microsomes suggested that pgp35 (SSR alpha) and calnexin were major Ca(2+)-binding proteins of the endoplasmic reticulum membrane. We propose that the function of the complex is to regulate Ca(2+)-dependent retention mechanisms for luminal proteins of the endoplasmic reticulum.

PMID:
1918067
[PubMed - indexed for MEDLINE]
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