Previous experiments suggested that trafficking of the a-factor transporter Ste6 of Saccharomyces cerevisiae to the yeast vacuole is regulated by ubiquitination. To define the ubiquitinationdependent step in the trafficking pathway, we examined the intracellular localization of Ste6 in the ubiquitination-deficient doa4 mutant by immunofluorescence experiments, with a Ste6-green fluorescent protein fusion protein and by sucrose density gradient fractionation. We found that Ste6 accumulated at the vacuolar membrane in the doa4 mutant and not at the cell surface. Experiments with a doa4 pep4 double mutant showed that Ste6 uptake into the lumen of the vacuole is inhibited in the doa4 mutant. The uptake defect could be suppressed by expression of additional ubiquitin, indicating that it is primarily the result of a lowered ubiquitin level (and thus of reduced ubiquitination) and not the result of a deubiquitination defect. Based on our findings, we propose that ubiquitination of Ste6 or of a trafficking factor is required for Ste6 sorting into the multivesicular bodies pathway. In addition, we obtained evidence suggesting that Ste6 recycles between an internal compartment and the plasma membrane.

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Whereas oral nickel administration to C57BL/6 mice (Ni(high) mice) renders the animals tolerant to immunization with NiCl2 combined with H2O2 as adjuvant, as determined by ear-swelling assay, it fails to tolerize Jalpha18-/- mice, which lack invariant NKT (iNKT) cells. Our previous work also showed that Ni(high) splenic B cells can adoptively transfer the nickel tolerance to untreated (Ni(low)) recipients, but not to Jalpha18-/- recipients. In this study, we report that oral nickel administration increased the nickel content of splenic Ni(high) B cells and up-regulated their Fas expression while down-regulating expression of bcl-2 and Bcl-xL, thus giving rise to an Ag-carrying, apoptosis-prone B cell phenotype. Although oral nickel up-regulated Fas expression on B cells of both wild-type Ni(high) and Jalpha18-/- Ni(high) mice, only the former showed a reduced number of total B cells in spleen when compared with untreated, syngeneic mice, indicating that iNKT cells are involved in B cell homeostasis by eliciting apoptosis of effete B cells. Upon transfer of Ni(high) B cells, an infectious spread of nickel tolerance ensues, provided the recipients are immunized with NiCl2/H2O2. As a consequence of immunization, Fas ligand-positive (FasL+) iNKT cells appeared in the spleen and apparently elicited apoptosis of Ni(high) B cells. The apoptotic Ni(high) B cells were taken up by splenic dendritic cells, which thereby became tolerogenic for nickel-reactive Ni(low) T cells. In conclusion, FasL+ iNKT cells may act as ready-to-kill sentinels of innate immunity, but at the same time assist in tolerance induction by eliciting Fas/FasL-mediated apoptosis of effete, Ag-containing B cells.