Productive folding of tyrosinase ectodomain is controlled by the transmembrane anchor.
Transmembrane domains (TMDs) are known as structural elements required for the insertion into the membrane of integral membrane proteins. We have provided here an example showing that the presence of the TMD is compulsory for the productive folding pathway of a membrane-anchored glycoprotein. Tyrosinase, a type I transmembrane protein whose insertion into the melanosomal membrane initiates melanin synthesis, is misfolded and degraded when expressed as a truncated polypeptide. We used constructs of tyrosinase ectodomain fused with chimeric TMDs or glycosylphosphatidylinositol anchor to gain insights into how the TMD enables the productive folding pathway of the ectodomain. We found that in contrast to the soluble constructs, the membrane-anchored chimeras fold into the native conformation, which allows their endoplasmic reticulum exit. They recruit calnexin to monitor their productive folding pathway characterized by the post-translational formation of buried disulfides. Lacking calnexin assistance, the truncated mutant is arrested in an unstable conformation bearing exposed disulfides. We showed that the transmembrane anchor of a protein may crucially, albeit indirectly, control the folding pathway of the ectodomain.
Popescu
Costin I
CI
Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.
Mares
Alina
A
Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.
Zdrentu
Livia
L
Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania.
Zitzmann
Nicole
N
Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, OX1 3QU Oxford, United Kingdom.
Dwek
Raymond A
RA
Oxford Glycobiology Institute, Department of Biochemistry, South Parks Road, OX1 3QU Oxford, United Kingdom.
Petrescu
Stefana M
SM
Institute of Biochemistry, Splaiul Independentei 296, 060031 Bucharest 17, Romania. Electronic address: stefana.petrescu@biochim.ro.
D037281:Calnexin / Q000378:metabolism; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D006801:Humans; D008543:Melanins / Q000096:biosynthesis; D008565:Membrane Proteins / Q000737:chemistry*; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins
D017261:Glycosylphosphatidylinositols; D008543:Melanins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D037281:Calnexin; D014442:Monophenol Monooxygenase