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PA28 is a modulator of the 20S proteasome. The PA28 binding sites on the 20S proteasome are still not well defined. Using yeast two-hybrid interaction assays and proteasome inactivation kinetics we provide evidence that the proteasome α4 subunit is one of the PA28 binding sites. This finding is supported by the observation that a hepatitis Bvirus X protein-derived polypeptide habouring the α4 proteasome subunit binding motif impairs the activation of 20S proteasomes by PA28.