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First published online 25 May 2004
doi: 10.1242/jcs.01154

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Carbohydrates act as sorting determinants in ER-associated degradation of tyrosinase

¹ Department of Biochemistry and Molecular Biology, Program in Molecular and Cellular Biology, University of Massachusetts, 710 North Pleasant Street, Amherst, MA 01003, USA
² Department of Dermatology, Yale University School of Medicine, 15 York Street, New Haven, CT 06520, USA

View larger version (38K): [in a new window]   Fig. 1. Tyr and Tyr(C85S) are proteasome substrates. (A) Wild-type Tyr or mutant Tyr(C85S) mouse melanocytes were pulsed with [35S]Met/Cys for 30 minutes and chased for the indicated times. Lysates were immunoprecipitated with anti-tyrosinase antibodies. Arrowheads and arrows indicate high-mannose and complexed forms of tyrosinase, respectively. The plot displays the quantification of five separate experiments. (B) Tyr(C85S) melanocytes were treated as in A, except half of the samples were incubated with 25 µM lactacystin (LCT) during the starvation, pulse, and chase periods. The plot displays the quantification of four separate experiments. (C) Tyr(C85S) melanocytes were incubated for 4 hours in the presence of various inhibitors: DMSO, 50 µM Leu-Leu nor-Leucinal (LLnL), 25 µM LCT, 50 µM MG132, or 50 µM E64 as indicated. Cell lysates were then either directly subjected to immunoblotting using anti-tyrosinase antibodies, or fractured and ultracentrifuged to separate the cytosol (S) from the membrane (P). Unglycosylated tyrosinase is indicated by a double arrowhead.

View larger version (32K): [in a new window]   Fig. 2. Degradation of albino TYR(C89R) can be reconstituted with semi-permeabilized melanocytes. (A) TYR or TYR(C89R) mRNAs were translated in the presence of Tyr(C85S) or wild-type Tyr SP-melanocytes (membranes), or microsomes (MS). Protein synthesis was terminated by the addition of cycloheximide and MS or SP-melanocytes were isolated. Isolated membranes were resuspended in an Energy Regeneration System (ERS) in Untreated rabbit Reticulocyte Lysate (URL), and incubated at 37°C for the indicated time. TYR and UTYR indicate translocated and untranslocated tyrosinase, respectively. (B) TYR(C89R) mRNA was translated in RRL in the presence of Tyr(C85S) SP cells and either directly incubated with either URL or KHM buffer devoid of proteasomes. All the samples were incubated at 37°C for the indicated time and treated as above. (C) Proteins from Tyr(C85S) cell lysates (lane 1) or SP-melanocytes incubated in the presence of buffer (lane 2) or URL (lane 3) were separated by SDS-PAGE, and subjected to immunoblotting with antibodies to the -subunit of the proteasome.

View larger version (37K): [in a new window]   Fig. 3. Mannose and glucose trimming play a role in the degradation of Tyr(C85S). (A) Diagram of the specific effects of the glucose and mannose analogues, n-butyl deoxynanojirimycin (DNJ) and deoxymannojirimycin (DMJ) on the trimming of the Glc3Man9GlcNAc2 carbohydrate moiety. Triangles, circles, and squares indicate glucose, mannose, and N-acetyl-glucosamine residues, respectively. (B) Pulse-chase analysis of Tyr(C85S) mouse melanocytes incubated with DNJ as indicated and/or with DMJ throughout starvation, pulse and chase periods, except for samples designated with an asterisk which indicates post-translational addition. 0 and 3 hour time points are indicated in the graph as filled and open bars, respectively. (C) TYR(C89R) mRNA was translated in the presence Tyr(C85S) SP-melanocytes and resuspended in URL or buffer as described previously with or without DMJ and DNJ as indicated.

View larger version (37K): [in a new window]   Fig. 4. Loss of triglucosylated Tyr(C85S) is unaffected by known protease inhibitors. (A) Pulse chase analysis of Tyr(C85S) or (B) wild-type Tyr in mouse melanocytes in the presence or absence of the indicated inhibitors throughout the time course, while an asterisk indicates the post-translational addition of the inhibitor. LP indicates leupeptin and pepstatin (10 µg/ml). (C) Human TYR(C89R) was translated in the presence of Tyr(C85S) SP-cells, with or without DNJ and/or ß-lac and separated on SDS-PAGE as previously described.

View larger version (72K): [in a new window]   Fig. 5. Albino Tyr(C85S) forms aggregates in the absence of glucose trimming. After a 1-hour incubation in the presence or absence of DNJ, Tyr and Tyr(C85S) melanocytes were lysed in 2% CHAPS, separated on a 5-25% linear sucrose gradient by ultracentrifugation, and subjected to immunoblotting as previously described. The stacking gels were included in the autoradiography.

View larger version (28K): [in a new window]   Fig. 6. Triglucosylated TYR(C89R) is degraded by the proteasome at a rate similar to the control. (A) TYR(C89R) was translated in the presence of SP-melanocytes with or without DNJ and/or ß-lac for 1 hour. Isolated membranes were resuspended in URL and chased at 37°C. Samples were removed at the indicated chase times, split and either separated on SDS-PAGE as described previously, or precipitated with TCA. The resulting supernatants were quantified using a scintillation counter. (B) Tyr(C85S) melanocytes were incubated with DNJ in the presence or absence of LCT, and chased with the protein synthesis inhibitor cycloheximide for 8 hours. Melanocytes were then fixed, permeabilized, and labeled with anti-tyrosinase or anti-BiP antibodies.

View larger version (45K): [in a new window]   Fig. 7 . BiP and PDI, but not calnexin, colocalize with aggregates of triglucosylated Tyr(C85S). (A) After a 4-hour incubation in the presence or absence of 500 µM DNJ, wild-type Tyr and (B) mutant Tyr(C85S) mouse melanocytes were fixed, permeabilized and labeled with anti-tyrosinase (green), anti-BiP, anti-PDI, or anti-calnexin antibodies (red) as indicated. Colocalization is indicated by yellow coloration. Bar, 2 µm.

View larger version (37K): [in a new window]   Fig. 8. Aggregation of TYR(C89R) is inhibited by reducing agent and induced by non-hydrolyzable ATP. (A) TYR(C89R) was translated in the presence of SPTyr(C85S) melanocytes for 1 hour with or without DNJ or DTT. Membranes were isolated and resuspended in a proteasome-free buffer that contained DNJ, ATP--S or DTT. Samples were isolated at sequential time points and either separated on SDS-PAGE or precipitated with TCA (B).