AP-1 and AP-3 are present on distinct endosomal buds in MNT-1 cells. Whole-mount preparations (A and B) and ultrathin cryosections (C-F) of MNT-1 cells were double immunogold labeled as indicated using PAG10 and PAG15. Before immunogold labeling, cells were incubated at 37°C for 1 h with Tf-HRP and then processed for whole-mount cytochemistry (A and B) or with Tf-FITC and then fixed and cryosectioned (E, E inset, and F). Arrowheads indicate labeling for AP-3 (A, B, and D) or Hrs (C). Arrows indicate labeling for AP-1 (A-D) or buds labeled for both AP-1 and Tf-FITC (E and F). Note the predominant labeling for AP-1 near stage IV melanosomes (IV) in A-E and the bud labeled for both AP-1 and AP-3 (B, inset). Bars, 200 nm.

HRP-tyrosinase chimera lacking the dileucine-based signal accumulates downstream of tubular endosomes. MNT-1 cells were transiently transfected with HRP-tyrAA and processed for HRP cytochemistry and Epon embedding. (A) Accumulation of HRP-tyrAA in tubular and vacuolar compartments often showing associated buds (arrows). Note the absence of accumulation of HRP-tyrAA in the Golgi area (arrowheads). (B) HRP-tyrAA is also detected at the plasma membrane (PM) and arrows. (C) HRP-tyrAA is present in compartments with features of MVBs (star) but not in the Golgi (arrowheads) or in melanosomes (arrows). III, stage III melanosomes; GA, Golgi apparatus. Bars, 200 nm.

Tyrosinase accumulates in AP-3- and AP-1-enriched endosomal domains. Ultrathin cryosections of melan-a cells were double labeled for tyrosinase (PAG10) and either AP-3 (PAG15; A-C) or AP-1 (PAG15; D). (A and B) Note the AP-3-positive vesicles and tubules containing tyrosinase (arrows) that are close to stage IV melanosomes (A, B, and B inset). (C) Tyrosinase near the Golgi (GA) is found in vesicles that lack apparent coats (arrows) and only infrequently label for AP-3 (C, arrowhead). (D) Tyrosinase and AP-1 coincide in coated vesicles and tubules (arrows). Bars, 200 nm.

Increased association of tyrosinase with AP-1 in AP-3-deficient melanocytes. Ultrathin cryosections of melan-pe cells were immunogold labeled for tyrosinase alone (PAG10; A) or with AP-1 (PAG15; B and C). Note that stage III and IV melanosomes are present within melan-pe cells and that tyrosinase labeling can be observed on them (A and C) in addition to the vacuolar endosomes (star). Coincidence of labeling for AP-1 and tyrosinase (B and C, arrows) is observed at high levels on coated tubules and vesicles near vacuolar endosomes (B, star) and melanosomes (C, IV). Bars, 200 nm.

AP-3 localizes to buds near endosomes and melanosomes in MNT-1 cells. (A) Ultrathin cryosections of MNT-1 cells were immunogold labeled for AP-3 (PAG15) or AP-3 (PAG15) and Hrs (PAG10). A region near a stage IV melanosome is shown. Arrows highlight AP-3 labeling in buds and tubules closely apposed to the melanosome. (B, C, and E) MNT-1 cells were incubated at 37°C for 1 h with Tf-HRP and then processed for whole-mount cytochemistry. Cells were immunogold labeled for AP-3 (PAG15) and Hrs (PAG10; B), the cytoplasmic domain of Pmel17 (PAG10; C), or the clathrin light chain (PAG10; E). Arrows point to clusters of AP-3 labeling on endosomal buds closely apposed to melanosomes (B) and linked to electron-dense endosomes that label for Pmel17 (C) and Hrs (B). The AP-3-labeled buds also label for clathrin (E). (D) Ultrathin cryosection of MNT-1 cells incubated at 37°C for 1 h with Tf-FITC and double labeled for AP-3 (PAG10) and FITC (PAG5). Note the continuity of the AP-3 labeled bud to FITC labeled tubules. Bars, 200 nm.

AP-1 and AP-3 bind to the dileucine-based targeting signal of tyrosinase. (A) Sequences of the cytoplasmic domains of mouse tyrosinase, Tyrp1 and Tyrp2. Known or putative dileucine- and tyrosine-based sorting signals and amino acid numbers are indicated. (B) The indicated cytoplasmic domains fused to the Gal4BD were tested in a yeast three-hybrid assay with the Gal4AD fused to the indicated AP heavy chains (γ1 for AP-1, αC for AP-2, δ for AP-3, or ε for AP-4) in the absence or presence of the cognate light chains (σ1A, σ2, σ3A, σ3B, or σ4). S. cerevisiae HF7c cells transduced with the indicated plasmids were patched onto minimal medium plates with (+His) or without (-His) histidine. Growth in the absence of histidine indicates a positive interaction. (C) Yeast three-hybrid analysis of tyrosinase cytoplasmic domain constructs, fused to the Gal4BD, coexpressed with the σ1A and Gal4AD fused to either γ1 or αC. S. cerevisiae HF7c cells transduced with all three plasmids were grown in minimal medium plates with (+His) or without (-His) histidine. The tyrosinase fusions were either WT or site-directed mutants of the indicated leucine residues to alanine. (D) GST or GST fusion proteins containing the WT tyrosinase cytoplasmic domain or site-directed mutants with alanine substitutions at the relevant (L517 and 518A) or irrelevant (L527 and 528A) dileucine sequences were incubated with bovine brain extracts and then precipitated with glutathione-Sepharose. The precipitates were fractionated by SDS-PAGE and probed by immunoblotting with antibodies to the γ1 or σ3 chains of AP-1 and AP-3, respectively. The last lane is a percentage of the input extract for the precipitations. Positions of the relevant chains are noted to the left; σ3 migrates as a doublet.

Tyrosinase localizes to tubular structures near endosomal compartments in addition to melanosomes and vesicles in the Golgi area. (A-D) Ultrathin cryosections of melan-a cells were immunogold labeled for tyrosinase with PAG10. (A and B) Tyrosinase labeling the limiting membrane of endosomes and stage III and IV melanosomes. Stage II premelanosomes with characteristic internal striations but no pigment (II) are not labeled. (B) A section emphasizing the Golgi area (GA). Note tyrosinase labeling in Golgi stacks and predominantly uncoated vesicles near the TGN (arrows). (C) Prominent tyrosinase labeling is detected on tubules (arrows) closely apposed to vacuolar endosomes (star). (D) Tyrosinase labeling of tubules (arrows) closely apposed to stage IV melanosomes. Bars, 200 nm.

(facing page). Tyrosinase accumulates in endosomal structures in AP-3-deficient melanocytes. (A and B) Immunoblot analysis of tyrosinase (Tyr; A) and AP-3 subunits δ, β3A, and μ3 (B) in melan-a (a) and melan-pe (pe) cells. Tyrosinase (A) or AP-3 (B) was immunoprecipitated from Triton X-100 lysates of equal numbers of melan-a and melan-pe cells and then fractionated by SDS-PAGE and immunoblotted with the indicated antibodies. Numbers indicate positions of molecular weight markers. (C and D) Ultrathin cryosections of melan-pe cells were double labeled for tyrosinase (PAG15) and Hrs (PAG10) (C) or single labeled for tyrosinase (D). Note the accumulation of tyrosinase on MVBs (stars) and on vacuolar endosomes (arrowheads) labeled for Hrs (arrows in C, inset). (E and F) Melan-pe cells were analyzed by DOPA cytochemistry for tyrosinase activity. Note the accumulation of reaction product in vacuolar endosomes (E and F; see star in F) and tubules associated with these structures (arrows, F) and the Golgi (GA) (E). (G) Melan-pe cells immunogold labeled for tyrosinase with PAG15, emphasizing labeling on vacuolar endosomes (star), associated tubules, and coated vesicles (arrows). Bars, 200 nm.