Locating Polyubiquitin Receptors on the 19S Regulatory Proteasome of <i>S. cerevisiae</i> 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 <i>S. cerevisiae</i> 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.