CHARACTERIZATION OF 2 POLYUBIQUITIN BINDING-SITES IN THE 26-S PROTEASE SUBUNIT 5A

Ubiquitylated proteins are degraded by the 26 S protease, an enzyme co mplex that contains 30 or more unique subunits, One of these proteins, subunit 5a (S5a), has been shown to bind ubiquitin-lysozyme conjugate s and free polyubiquitin chains, Using deletional analysis, we have id entified in the carboxyl-terminal half of human S5a, two independent p olyubiquitin binding sites whose sequences are highly conserved among higher eukaryotic S5a homologs, The sites are approximately 30-amino a cids long and are separated by 50 intervening residues, When expressed as small fragments or when present in full-length S5a molecules, the sites differ at least 10-fold in their apparent affinity for polyubiqu itin chains, Each binding site contains 5 hydrophobic residues that fo rm an alternating pattern of large and small side chains, e.g. Leu-Ala -Leu-Ala-Leu, and this pattern is essential for binding ubiquitin chai ns, Based on the importance of the alternating hydrophobic residues in the binding sites and previous studies showing that a hydrophobic pat ch on the surface of ubiquitin is essential for proteolytic targeting, we propose a model for molecular recognition of polyubiquitin chains by S5a.