A Common Temperature-sensitive Allelic Form of Human Tyrosinase Is Retained in the Endoplasmic Reticulum at the Nonpermissive Temperature

Abstract

Oculocutaneous albinism type 1TS is caused by mutations that render the melanocyte-specific enzyme tyrosinase temperature-sensitive (ts); the enzyme is inactive in cells grown at 37°C but displays full activity in cells grown at 31°C. To distinguish whether the ts phenotype of the common R402Q variant of human tyrosinase is due to altered enzymatic activity or to misfolding and a defect in intracellular trafficking, we analyzed its localization and processing in transiently transfected HeLa cells. R402Q tyrosinase accumulates in the endoplasmic reticulum (ER) at 37°C but exits the ER and accumulates in endosomal structures in cells grown at 31°C. The inability of the R402Q variant to exit the ER is confirmed by the failure to acquire endoglycosidase H resistance at 37°C and cannot be accounted for solely by enhanced proteasome-mediated degradation. ER retention at 37°C is mediated by the lumenal domain of R402Q tyrosinase, is not dependent on tethering to the membrane, and is irreversible. Finally, a wild-type allelic form of tyrosinase is partially ts in transiently transfected HeLa cells. The data show that human tyrosinase expressed in non-melanogenic cells folds and exits the ER inefficiently and that R402Q tyrosinase exaggerates this defect, resulting in a failure to exit the ER at physiologic temperatures.

Pink-eyed Dilution Protein Controls the Processing of Tyrosinase

“…Most Tyr mutations examined to date are associated with ER retention, rather than loss of Tyr activity as was predicted previously. The temperature-sensitive Tyr mutant R402Q can exit the ER at 31°C, but accumulates in the ER at 37°C (Berson et al, 2000). Thus, proper processing of Tyr in the ER is vital to its enzymatic activities and subcellular localization.…”

“…Endoplasmic reticulum (ER) processing of Tyr requires the presence of the chaperone calnexin, which is thought to increase ER retention time for Tyr Toyofuku et al, 1999). The most common Tyr mutations are associated with ER retention of the protein, presumably the result of misfolding (Berson et al, 2000;Halaban et al, 2000b;Toyofuku et al, 2001). In addition, substrates DOPA and tyrosine promote Tyr exit from the ER and are associated with induction of Tyr activity and melanin synthesis in amelanotic melanoma cells (Halaban et al, 2000a).…”

Structure and Function of Human Tyrosinase and Tyrosinase‐Related Proteins

“…As ubset of OCA1 relatedm utations, including R402Q, [32] P406L, [33] and R422Q, [34] causes temperature-sensitive oculocutaneous albinism (OCA1-TS), due to at emperature-sensitive form of TYR with optimal tyrosinase activity at temperatures below 37 8C. As aresult,pigmentation is generally more prominent in the extremities (ears, face and legs), where the temperature is lower than in other parts of the body.I nt he TYR model( Figure 6) R402 is surface-exposed and forms ah ydrogen bond with Q399.…”

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…Metabolic labeling of MNT-1 cells or transiently transfected HeLa cells and immunoprecipitation from Triton X-100 cell lysates were done essentially as described previously (Berson et al, 2000) by using EXPRE 35 S 35 S 35 S-cysteine/ methionine mixture (PerkinElmer Life and Analytical Sciences, Boston, MA). Cells were labeled for 30 min at 37°C and chased for the indicated times.…”