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 K_d = 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.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an open state, while Cse4 Y193A predominantly exhibits a closed state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an open state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

A surprising complexity of ubiquitin signaling has emerged with identification of different ubiquitin chain topologies. However, mechanisms of how the diverse ubiquitin codes control biological processes remain poorly understood. Here, we use quantitative whole-proteome mass spectrometry to identify yeast proteins that are regulated by lysine 11 (K11)-linked ubiquitin chains. The entire Met4 pathway, which links cell proliferation with sulfur amino acid metabolism, was significantly affected by K11 chains and selected for mechanistic studies. Previously, we demonstrated that a K48-linked ubiquitin chain represses the transcription factor Met4. Here, we show that efficient Met4 activation requires a K11-linked topology. Mechanistically, our results propose that the K48 chain binds to a topology-selective tandem ubiquitin binding region in Met4 and competes with binding of the basal transcription machinery to the same region. The change to K11-enriched chain architecture releases this competition and permits binding of the basal transcription complex to activate transcription.

Understanding the normal function of the Huntingtin (HTT) protein is of significance in the design and implementation of therapeutic strategies for Huntington's disease (HD). Expansion of the CAG repeat in the HTT gene, encoding an expanded polyglutamine (polyQ) repeat within the HTT protein, causes HD and may compromise HTT's normal activity contributing to HD pathology. Here, we investigated the previously defined role of HTT in autophagy specifically through studying HTT's association with ubiquitin. We find that HTT interacts directly with ubiquitin in vitro. Tandem affinity purification was used to identify ubiquitinated and ubiquitin-associated proteins that copurify with a HTT N-terminal fragment under basal conditions. Copurification is enhanced by HTT polyQ expansion and reduced by mimicking HTT serine 421 phosphorylation. The identified HTT-interacting proteins include RNA-binding proteins (RBPs) involved in mRNA translation, proteins enriched in stress granules, the nuclear proteome, the defective ribosomal products (DRiPs) proteome and the brain-derived autophagosomal proteome. To determine whether the proteins interacting with HTT are autophagic targets, HTT knockout (KO) cells and immunoprecipitation of lysosomes were used to investigate autophagy in the absence of HTT. HTT KO was associated with reduced abundance of mitochondrial proteins in the lysosome, indicating a potential compromise in basal mitophagy, and increased lysosomal abundance of RBPs which may result from compensatory up-regulation of starvation-induced macroautophagy. We suggest HTT is critical for appropriate basal clearance of mitochondrial proteins and RBPs, hence reduced HTT proteostatic function with mutation may contribute to the neuropathology of HD.