Translation rate of human tyrosinase determines its N-linked glycosylation level.
Tyrosinase is a type I membrane glycoprotein essential for melanin synthesis. Mutations in tyrosinase lead to albinism due, at least in part, to aberrant retention of the protein in the endoplasmic reticulum and subsequent degradation by the cytosolic ubiquitin-proteasomal pathway. A similar premature degradative fate for wild type tyrosinase also occurs in amelanotic melanoma cells. To understand critical cotranslational events, the glycosylation and rate of translation of tyrosinase was studied in normal melanocytes, melanoma cells, an in vitro cell-free system, and semi-permeabilized cells. Site-directed mutagenesis revealed that all seven N-linked consensus sites are utilized in human tyrosinase. However, glycosylation at Asn-290 (Asn-Gly-Thr-Pro) was suppressed, particularly when translation proceeded rapidly, producing a protein doublet with six or seven N-linked core glycans. The inefficient glycosylation of Asn-290, due to the presence of a proximal Pro, was enhanced in melanoma cells possessing 2-3-fold faster (7.7-10.0 amino acids/s) protein translation rates compared with normal melanocytes (3.5 amino acids/s). Slowing the translation rate with the protein synthesis inhibitor cycloheximide increased the glycosylation efficiency in live cells and in the cell-free system. Therefore, the rate of protein translation can regulate the level of tyrosinase N-linked glycosylation, as well as other potential cotranslational maturation events.
Ujvari
A
A
Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003, USA.
Aron
R
R
Eisenhaure
T
T
Cheng
E
E
Parag
H A
HA
Smicun
Y
Y
Halaban
R
R
Hebert
D N
DN
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D016384:Consensus Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008544:Melanocytes / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D014442:Monophenol Monooxygenase / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021381:Protein Transport
D008562:Membrane Glycoproteins; D014442:Monophenol Monooxygenase