Thus, the transport pathways in which AP-3 functions are likely redundant in most cell types but necessary for unique events in certain cells. unique endosomal subdomains to melanosomes and that the AP-3 pathway ensures that tyrosinase averts entrapment on internal membranes of forming multivesicular bodies. INTRODUCTION Sorting of integral membrane proteins R428 among post-Golgi organelles is usually facilitated by cytoplasmic coats, including the ubiquitously expressed, heterotetrameric adaptor protein (AP) R428 complexes. By binding cytoplasmic sorting signals on cargo proteins, AP complexes recruit cargo to patches on donor membranes that bud to form vesicles or tubules destined to fuse with target membranes (Bonifacino and Traub, 2003 Jun ; Robinson, 2004 ). Genetic analyses have shown that whereas deficiency of AP-1 or AP-2 results in embryonic lethality, AP-3 deficiency is not lethal and results in severe phenotypes only in specific tissues (examined in Boehm and Bonifacino, 2002 ). Thus, the transport pathways in which AP-3 functions are likely redundant in most cell types but necessary for unique events in certain cells. Defining these pathways and those involving the essential APs will be necessary to fully understand endosomal maturation and the formation of tissue-specific organelles. How and where AP-3 functions in nonspecialized cells is usually controversial. Yeast AP-3 binds to cytoplasmic dileucine-like sorting signals of cargo, such as alkaline phosphatase and Vam3p, and facilitates their biosynthetic traffic to the vacuole in a pathway that bypasses the prevacuolar compartment, analogous to the mammalian multivesicular late endosome/multivesicular body (MVB) (examined in Burd (pearl) mice (The Jackson Laboratory, Bar Harbor, ME) according to published protocols (Sviderskaya S-Cells were fixed with 2% (wt/vol) paraformaldehyde (PFA) or with a mixture of 2% (wt/vol) PFA and 0.2% (wt/vol) glutaraldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Cells were processed for ultracryomicrotomy as explained previously (Raposo Cells were washed with serum-free medium and R428 starved for 45 min before incubation with Tf-FITC (60 g/ml) for 45 min at 37C. After washing with ice-cold medium, cells were fixed and processed for ultracryomicrotomy. The relative distribution of AP-3 and AP-1 in melan-a cells was evaluated by analyzing directly under the electron microscope randomly selected cell profiles from two unique grids. In total, 450 and 448 platinum particles were counted for AP-3 and AP-1, respectively, and assigned to the compartment over which they were located. The definition of the unique compartments was based on their morphology and their previous characterization by immunogold labeling with different organelle markers (EEA-1 and Hrs for early endosomes; TGN38 and TGN46 for the TGN; and LAMP-1 for late endosomes/lysosomes) and internalization of endocytic tracers (bovine serum albumin-gold and Tf-FITC). Tubulovesicular membranes that were located at the Immunogold labeling of AP-3, AP-1, Clathrin, Hrs, Pmel17 (using Pep13h antibody to the cytoplasmic domain name), and Tf receptor on whole-mounted cells was performed as explained previously (Stoorvogel MNT-1 cells transfected with HRP-tyr and HRP-tyrAA for 24 or 48 h were fixed with 2% PFA/0.5% glutaraldehyde in 0.2 M PB, pH 7.4, for 90 min. After several washes with 50 mM Tris-HCl, pH 7.6, cells were treated for 20 min with 0.03% DAB in the presence of 1 l/ml H2O2 (30 vol). Cells were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 90 min, postfixed with 2% OsO4, dehydrated in ethanol, and embedded in Epon. Ultrathin sections were counterstained with uranyl acetate before observation. DOPA histochemistry was carried out as explained previously (Boissy Golgi TGN TVEs/End TVEs/MVBs TVEs/Melan TVEs/Lys Vesicles/cytoplasm AP-3 3.5 9.4 41.5 4 25.8 1.8 14 AP-1 0 17.6 39 4 19 1.5 18.5 Open in a separate window TVEs, tubulovesicular elements close to endosomal vacuoles (End), melanosomes (Melan), MVBs, and lysosomes (Lys). Figures symbolize the percentages of platinum particles labeling AP-3 and AP-1 over.