Folding and maturation of tyrosinase-related protein-1 are regulated by the post-translational formation of disulfide bonds and by N-glycan processing.
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.
Negroiu
G
G
Institute of Biochemistry of the Romanian Academy, Splaiul Independentei 296, 77700 Bucharest, Romania.
Dwek
R A
RA
Petrescu
S M
SM
D017485:1-Deoxynojirimycin / Q000031:analogs & derivatives / Q000494:pharmacology; D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D037281:Calnexin; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D065089:Glycoside Hydrolase Inhibitors; D006596:Hexosaminidases / Q000378:metabolism; D007700:Kinetics; D008545:Melanoma / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D010088:Oxidoreductases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014407:Tumor Cells, Cultured; D000520:alpha-Glucosidases / Q000378:metabolism
D002135:Calcium-Binding Proteins; D004220:Disulfides; D065089:Glycoside Hydrolase Inhibitors; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D011134:Polysaccharides; D011506:Proteins; D037281:Calnexin; D017485:1-Deoxynojirimycin; C059896:miglustat; D010088:Oxidoreductases; C079621:Tyrp1 protein, mouse; C064056:tyrosinase-related protein-1; D006596:Hexosaminidases; D000520:alpha-Glucosidases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D004229:Dithiothreitol