Folding and maturation of tyrosinase-related protein-1 are regulated by the post-translational formation of disulfide bonds and by N-glycan processing

J Biol Chem. 2000 Oct 13;275(41):32200-7. doi: 10.1074/jbc.M005186200.

Abstract

In this study we have explored the endoplasmic reticulum associated events accompanying the maturation of the tyrosinase-related protein-1 (TRP-1) nascent chain synthesized in mouse melanoma cells. We show that TRP-1 folding process occurs much more rapidly than for tyrosinase, a highly homologous protein, being completed post-translationally by the formation of critical disulfide bonds. In cells pretreated with dithiothreitol (DTT), unfolded TRP-1 is retained in the endoplasmic reticulum by a prolonged interaction with calnexin and BiP before being targeted for degradation. The TRP-1 chain was able to fold into DTT-resistant conformations both in the presence or absence of alpha-glucosidase inhibitors, but folding occurred through different pathways. During the normal folding pathway, TRP-1 interacts with calnexin. In the presence of alpha-glucosidase inhibitors, the interaction with calnexin is prevented, with TRP-1 folding being assisted by BiP. In this case, the process has similar kinetics to that of untreated TRP-1 and yields a compact form insensitive to DTT as well. However, this form has different thermal denaturation properties than the native conformation. We conclude that disulfide bridge burring is crucial for the TRP-1 export. This suggests that although various folding pathways may complete this process, the native form may be acquired only through the normal unperturbed pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Deoxynojirimycin / analogs & derivatives
  • 1-Deoxynojirimycin / pharmacology
  • Amidohydrolases / metabolism
  • Animals
  • Calcium-Binding Proteins / metabolism
  • Calnexin
  • Disulfides / chemistry
  • Disulfides / metabolism*
  • Dithiothreitol / pharmacology
  • Endoplasmic Reticulum / enzymology
  • Endoplasmic Reticulum / metabolism
  • Glycoside Hydrolase Inhibitors
  • Hexosaminidases / metabolism
  • Kinetics
  • Melanoma / metabolism
  • Melanoma / pathology
  • Membrane Glycoproteins*
  • Mice
  • Molecular Chaperones / metabolism
  • Oxidoreductases*
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Polysaccharides / metabolism*
  • Protein Binding
  • Protein Conformation / drug effects
  • Protein Denaturation / drug effects
  • Protein Folding*
  • Protein Processing, Post-Translational* / drug effects
  • Protein Transport / drug effects
  • Proteins / chemistry*
  • Proteins / metabolism*
  • Thermodynamics
  • Tumor Cells, Cultured
  • alpha-Glucosidases / metabolism

Substances

  • Calcium-Binding Proteins
  • Disulfides
  • Glycoside Hydrolase Inhibitors
  • Membrane Glycoproteins
  • Molecular Chaperones
  • Polysaccharides
  • Proteins
  • Calnexin
  • 1-Deoxynojirimycin
  • miglustat
  • Oxidoreductases
  • Tyrp1 protein, mouse
  • tyrosinase-related protein-1
  • Hexosaminidases
  • alpha-Glucosidases
  • Amidohydrolases
  • Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase
  • Dithiothreitol