Locating Polyubiquitin Receptors on the 19S Regulatory Proteasome of S. cerevisiae by Cross-Linking Mass Spectrometry.

The effectiveness of state-of-the-art cross-linking strategies and mass spectrometry (MS) detection was explored in an important biological context, namely, the ubiquitin-proteasome system, which is responsible for most of the regulated protein degradation in eukaryotic cells. The locations of possible binding sites on the S. cerevisiae 19S proteasome regulatory particle for Lys48 linked polyubiquitin chains were examined using cross-linking strategies and MS based detection by comparing two types of cross-linkers: a (bis)-sulfosuccinimidyl suberate (BS3) and diethyl suberothioimidate (DEST). The well-established BS3-based strategy produced 328 cross-linked peptides; however, no ubiquitin-19S cross-links were observed. The recently developed DEST-based approach produced fewer (146) linkages overall, but these included six ubiquitin-19S cross-links. Some of these cross-links are predicted by the canonical view of ubiquitin recognition, but others suggest novel insights into how the proteasome recognizes its substrates. A discussion of these strategies and structural implications for polyubiquitin-proteasome binding is provided.