Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation

Mol Cell Biol. 1992 Jul;12(7):3288-96. doi: 10.1128/mcb.12.7.3288.


SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Base Sequence
  • Biological Transport, Active
  • DNA Mutational Analysis
  • Endoplasmic Reticulum / chemistry
  • Endoplasmic Reticulum / metabolism
  • Escherichia coli Proteins
  • Fluorescent Antibody Technique
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism*
  • HSP40 Heat-Shock Proteins
  • Heat-Shock Proteins / genetics
  • Membrane Proteins / genetics*
  • Membrane Proteins / metabolism
  • Membrane Transport Proteins*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Sequence Homology, Nucleic Acid
  • Subcellular Fractions / chemistry


  • Bacterial Proteins
  • DnaJ protein, E coli
  • Escherichia coli Proteins
  • Fungal Proteins
  • HSP40 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Recombinant Fusion Proteins
  • SEC63 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins