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Acidic clusters act as sorting signals for packaging cargo into clathrin-coated vesicles (CCVs), and also facilitate down-regulation of MHC-I by HIV-1 Nef. To find acidic cluster sorting machinery, we performed a gene-trap screen and identified the medium subunit (µ1) of the clathrin adaptor AP-1 as a top hit. In µ1 knockout cells, intracellular CCVs still form, but acidic cluster proteins are depleted, although several other CCV components were either unaffected or increased, indicating that cells can compensate for long-term loss of AP-1. In vitro experiments showed that the basic patch on µ1 that interacts with the Nef acidic cluster also contributes to the binding of endogenous acidic cluster proteins. Surprisingly, µ1 mutant proteins lacking the basic patch and/or the tyrosine-based motif binding pocket could rescue the µ1 knockout phenotype completely. In contrast, these mutants failed to rescue Nef-induced down-regulation of MHC class I, suggesting a possible mechanism for attacking the virus while sparing the host cell.

Contribution of the clathrin adaptor AP-1 subunit µ1 to acidic cluster protein sorting