Promotion of tyrosinase folding in COS 7 cells by calnexin.
To understand the process of expression of tyrosinase, a key enzyme of melanogenesis, we examined its maturation in the endoplasmic reticulum (ER) by using a heterogeneous expression system. When human tyrosinase cDNA was introduced into COS 7 cells, tyrosinase activity was minimally detected. Immunofluorescence study revealed that tyrosinase was immunolocalized in the nuclear rim, the reticular network, and the punctuated structures. Because a cytoplasmic tail of tyrosinase-gene family protein functions as a lysosomal targeting signal in non-melanocytic cells, and immature and/or misfolded molecules are selectively retained in the ER, the observed localization suggested the inefficient maturation in the COS 7 cells. We thus examined if supplementation of calnexin, a membrane-bound chaperone with affinity for oligosaccharide-processing intermediates containing monoglucose, could improve the process. As expected, the activity was enhanced approximately 2-fold by co-transfection of cDNA encoding calnexin. In contrast, co-transfection of the cytosolic tail-free calnexin, which inhibits calnexin function by allowing premature egress of its ligands from the ER, suppressed expression of this enhanced tyrosinase activity. When alpha-glucosidase activity, which is required for calnexin function, was inhibited by castanospermine (CST) treatment, expression of tyrosinase activity was completely abolished. To confirm the direct involvement of calnexin in tyrosinase maturation, the interaction of calnexin with tyrosinase was examined. Immunoprecipitation of calnexin from extracts of [35S]methionine labeled cells with anti-calnexin antibody revealed that the association is highest immediately after the pulse and that nascent tyrosinase is gradually dissociated upon chase. The association was completely inhibited when CST was included in the medium. Hence, we suggest that the proper folding of tyrosinase is largely dependent on its direct interaction with calnexin for the determined duration in the ER.
Toyofuku
K
K
Division of Dermatology & Cutaneous Sciences, Faculty of Medicine, University of Alberta, T6G 2S2, Canada.
Wada
I
I
Hirosaki
K
K
Park
J S
JS
Hori
Y
Y
Jimbow
K
K
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D037281:Calnexin; D002240:Carbohydrate Sequence; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D008543:Melanins / Q000032:analysis* / Q000096:biosynthesis; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011233:Precipitin Tests; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008543:Melanins; D009844:Oligosaccharides; D011994:Recombinant Proteins; D037281:Calnexin; D014442:Monophenol Monooxygenase