THE HYDROPHOBIC EFFECT CONTRIBUTES TO POLYUBIQUITIN CHAIN RECOGNITION

The principal targeting signal used in the ubiquitin-proteasome degrad ation pathway is a homopolymeric, K48-linked polyubiquitin chain: the chain is recognized by a specific factor(s) in the 19S regulatory comp lex of the 26S proteasome, while the substrate is degraded by the 20S catalytic complex. We have previously presented evidence implicating t he side chains of L8, I44. and V70 in the recognition of K48-linked ch ains. In the crystal structure of tetraubiquitin, these side chains fo rm a repeating, surface-exposed hydrophobic patch. To test the hypothe sis that a close-packing interaction involving this patch is important for the chain recognition, residue 8 was mutated to a series of small er aliphatic amino acids (G, A, V). The effects of these mutations wer e first investigated in rabbit reticulocyte fraction II; even the seve rest truncating mutation (L8G) had only a modest inhibitory effect on the degradation of a model substrate (I-125-lactalbumin). We show that these steady-state degradation data substantially underestimate the d eleterious effects of these mutations on chain recognition by the prot easome, because the recognition step does not contribute to rate limit ation in the fraction II system. Much stronger inhibition was observed when chain binding was measured in a competition assay using purified 26S proteasomes, and the change in binding free energy depended linea rly on the surface area of the side chain. This behavior is consistent with a mode of binding in which the hydrophobic effect makes a favora ble contribution; i.e., one or more L8 side chains is shielded from so lvent when the chain binds to the 19S complex. A similar linear depend ence of binding energy an side chain area was observed for chain bindi ng to the 19S subunit known as S5a (as assayed using recombinant S5a b ound to nickel beads). Octa-ubiquitin (K-0.5 = 6 mu M) bound to S5a 4. 2-fold more tightly than tetra-ubiquitin; this is similar to the facto r of 5.8-fold relating the affinities of the same two chains for the 2 6S proteasome. Altogether, these findings indicate that the interactio n of K48-linked chains with the 19S complex is substantially similar t o the interaction of chains with isolated S5a. The results further sug gest that the hydrophobic patch is part of a minimum element which all ows for specific recognition of the polyubiquitin degradation signal b y the 26S proteasome.