Tyrosinase of albino melanocytes displays characteristics of ER-retained proteins. (A) The 70-kDa tyrosinase proteins are sensitive to endo H digestion. Western blots with anti-tyrosinase antibodies (T311 mAb, lanes 1–4; PEP7 rabbit polyclonal, lanes 5–12) showing nondigested (−) or endo H-digested (+) tyrosinase from WT human or mouse melanocytes, human albino (T373K), mouse melan c (C85S), or himalayan (H402A) melanocytes. The light and dark mouse himalayan melanocytes were collected from different flasks, all grown at 37°C; the difference in pigmentation was caused by yet undefined variability. Arrows indicate sizes of tyrosinase proteins in kDa: 80, mature; 70, immature; and 60, endo H digested. Molecular mass markers are designated in kDa by numbered bars on the left. (B) Immature tyrosinase associates with the ER chaperone calnexin and calreticulin. Western blots with anti-tyrosinase (T311 mAb, lanes 1–8; goat polyclonal, lanes 9–20) of WCL (40 μg protein/lane) from normal human or mouse melanocytes (WT), human albino (T373K), melan c (C85S), or himalayan (H402A), as indicated. Alternatively, cell lysates (250 μg protein/precipitation) were precipitated with rabbit polyclonal antibodies against calnexin (CNX), calreticulin (CRT), or rabbit IgG as a control (Cont). Immunoprecipitates then were subjected to Western blotting. The migration of tyrosinase in all of the coimmunoprecipitated reaction products is artificially retarded because of protein overloading contributed by the presence of high amounts of antibodies. (C) Association of newly synthesized tyrosinase with calnexin and calreticulin in WT and albino (C85S) melanocytes. Melanocytes were pulsed with [³⁵S]-Met/Cys for 30 min and then harvested immediately (lanes 1–3 and 7–9) or further incubated in media (without radioactive Cys/Met) for 2 h (lanes 4–6 and 10–12). Tyrosinase was precipitated directly with anti-tyrosinase antibodies (Tyr) or as an associated protein with anti-calnexin (CNX) or anti-calreticulin (CRT) antibodies. The latter were performed by sequential precipitation first with antibodies against the respective chaperone and, after dissociation of antibody-antigen complex, with a second immunoprecipitation with anti-tyrosinase antibodies. After resolution by SDS/PAGE, the levels of tyrosinase were quantified with a PhosphorImager.

Ectopic WT TYR-GFP chimeras display functions characteristic to tyrosinase. (A) Photograph of cell pellets showing pigmented WT mouse melanocytes (Host, WT), pigmented mutant melan c (Host, C85S) transfected with WT TYR-GFP (WT), or unpigmented melan c transfected with TYR(T373K)-GFP (T373K), compared with unpigmented nontransfected melan c melanocytes (none). There were approximately 30% and 80% fluorescing cells in the WT TYR-GFP and TYR(T373K)-GFP transfected melan c cultures. (B) Micrographs of WT mouse melanocytes transiently transfected with WT TYR-GFP. (Left) Green fluorescence from the fusion protein (GFP). (Middle) Red fluorescence of rhodamine-stained image detecting endogenous Tyr with PEP7 antibodies (Tyr). (Right) Merged image of the two. (The bar represents 10 μm.)

The albino T373K TYR mutation causes retention in the ER. Micrographs showing green fluorescence from the chimeric proteins WT TYR-GFP (WT) and TYR(T373K)-GFP (T373K) (Left), transiently expressed in WT mouse melanocytes, calnexin (CNX), and α-1,2-mannosidase II (Mann) stained with rhodamine (Middle), and merge of the two respective images (Right). (The bars represent 1 μm.)

Albino (C85S) mouse tyrosinase is retained in the ER. Immunofluorescence confocal microscopy of WT and albino (C85S) mouse melanocytes. (Left) The green fluorescein-stained panels represent tyrosinase detected with goat polyclonal anti-mouse tyrosinase antibodies. (Middle) The red rhodamine-stained panels show resident ER and Golgi complex markers, calnexin, and α-1,2-mannosidase II, respectively. (Right) Merged images. Tyrosinase colocalization with each protein is indicated in yellow in the merge images. (The bars represent 1 μm.)

Processing of ectopic TYR-GFP chimeras is similar to native proteins. (A) Western blots of TYR-GFP transfectants (40 μg protein/lane) grown continuously at 37°C or after a 14 h shift to 32°C. Host melanocytes were WT (Host WT, lanes 1–4 and 7–10) or albino (Host, C85S, lanes 5, 6, 11, and 12). The same membrane was first blotted with T311 mAb (lanes 1–6), and then, without stripping, with PEP7 (lanes 7–12). Melanocytes were harvested 1 week after transfection with WT TYR-GFP (WT), TYR-GFP(T373K) (T373K), or TYR(R402Q)-GFP (R402Q). Arrow indicates chimeric proteins of 100–110 kDa, and arrowhead points at endogenous Tyr. Bars indicate molecular mass protein markers. (B) Western blots with T311 mAb of undigested (−) and endo H-digested (+) proteins. Glycoproteins from extracts (300–400 μg/transfectant) were precipitated with wheat germ agglutinin-bound beads for 2 h and then incubated without (−) or with (+) endo H overnight. Arrow indicates the position of undigested chimeric proteins, gray arrowheads (in lanes 2 and 4) point to endo H-digested WT TYR-GFP proteins, and arrowheads mark endogenous Tyr. Host mouse melanocytes are as in A.

Exit of albino R402Q TYR from the ER is temperature dependent. TYR(R402Q)-GFP was transiently expressed in WT mouse melanocytes grown continuously at 37°C or shifted to the permissive temperature of 32°C for 14 h, as indicated. The micrographs show green fluorescence emitted from the chimeric protein TYR(R402Q)-GFP (Left), red fluorescence detecting calnexin (CNX) or α-1,2-mannosidase II (Mann) stained with rhodamine (Middle), and merged images (Right). (The bars represent 2 μm.)