Abstract
Protein ubiquitylation is an important posttranslational modification that governs most cellular processes. Signaling functions of ubiquitylation are very diverse and involve proteolytic as well as nonproteolytic events, such as localization, regulation of protein interactions, and control of protein activity. The intricacy of ubiquitin signaling is further complicated by several different polyubiquitin chain types that are likely recognized and interpreted by different protein readers. For example, K48-linked ubiquitin chains represent the most abundant chain topology and are the canonical degradation signals, but have been implicated in degradation-independent functions as well, likely requiring a variety of protein readers. Ubiquitin binding domains that interact with polyubiquitin chains are likely at the center of ubiquitin signal recognition and transmission, but their structure and selectivity are largely unexplored. Here we report identification and characterization of the ubiquitin interacting motif-like (UIML) domain of the yeast transcription factor Met4 as a strictly K48-polyubiquitin specific binding unit using methods such as biolayer interferometry (BLI), pull-down assays, and mass spectrometry. We further used the selective binding property to develop an affinity probe for purification of proteins modified with K48-linked polyubiquitin chains. The affinity probe has a Kd = 100 nM for K48 tetra-ubiquitin and shows no detectable interaction with either monoubiquitin or any other polyubiquitin chain configuration. Our results define a short strictly K48-linkage-dependent binding motif and present a new affinity reagent for the K48-polyubiquitin-modified proteome. Our findings benefit the ubiquitin field in analyses of the role of K48-linked polyubiquitylation and increase our understanding of chain topology selective ubiquitin chain recognition.
Highlights
Ubiquitylation is a post-translational modification that covalently attaches the small 76 residue ubiquitin protein to a lysine residue on target substrates
We have previously demonstrated by pull-down assays that the tandem UBD (tUBD) binds K48 ubiquitin chains starting at a chain length of 3 ubiquitins [39], and used K48-linked tetraubiquitin (4xUb(K48)) to characterize binding parameters of the tUBD and the individual ubiquitin interacting motif (UIM) and ubiquitin interacting motif like (UIML) domains, respectively (Figure 1)
This is in accordance with the results found in yeast cells where the Journal Pre-proof UIMLx2 probe primarily bound to K48 ubiquitin chains (Figure 4E)
Summary
Ubiquitylation is a post-translational modification that covalently attaches the small 76 residue ubiquitin protein to a lysine residue on target substrates. One of the first studied ubiquitin-binding protein, S5a/Rpn, was used to help in the enrichment of ubiquitinated proteins [34] This enrichment of ubiquitin modified proteins by UBDs eventually led researchers to develop tandem ubiquitin-binding entities (TUBEs), which was an arrangement of several UBD sequences engineered into the same amino acid chain [35]. These TUBEs were observed to have 100-1000 fold enhancement in Kd over a single UBD, they were used to help capture and purify all proteins that were modified by ubiquitin [28]. Journal Pre-proof In this report we characterize the tandem UBD of the transcription factor Met and find a strict K48 linkage dependence for poly-ubiquitin binding for the UIML domain. We used the ubiquitin chain selective part of the Met tandem UBD to engineer a K48 chain selective ubiquitin affinity reagent, which can be used to selectively enrich proteins modified with a K48-poly ubiquitin chain
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