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Genetic analysis reveals functions of atypical polyubiquitin chains.

ABSTRACT: Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in Saccharomyces cerevisiae. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R mutants import threonine poorly. The K11R mutant also exhibited a strong genetic interaction with a subunit of the anaphase-promoting complex (APC), suggesting a role in cell cycle regulation. K11-linkages are important for vertebrate APC function, but this was not previously described in yeast. We show that the yeast APC also modifies substrates with K11-linkages in vitro, and that those chains contribute to normal APC-substrate turnover in vivo. This study reveals comprehensive genetic interactomes of polyubiquitin chains and characterizes the role of K11-chains in two biological pathways.

SUBMITTER: Meza Gutierrez F

PROVIDER: S-EPMC6305200 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Genetic analysis reveals functions of atypical polyubiquitin chains.

Meza Gutierrez Fernando F Simsek Deniz D Mizrak Arda A Deutschbauer Adam A Braberg Hannes H Johnson Jeffrey J Xu Jiewei J Shales Michael M Nguyen Michelle M Tamse-Kuehn Raquel R Palm Curt C Steinmetz Lars M LM Krogan Nevan J NJ Toczyski David P DP

eLife 20181214

Although polyubiquitin chains linked through all lysines of ubiquitin exist, specific functions are well-established only for lysine-48 and lysine-63 linkages in <i>Saccharomyces cerevisiae</i>. To uncover pathways regulated by distinct linkages, genetic interactions between a gene deletion library and a panel of lysine-to-arginine ubiquitin mutants were systematically identified. The K11R mutant had strong genetic interactions with threonine biosynthetic genes. Consistently, we found that K11R ...[more]

PMID: 30547882

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Project description:Transactive response (TAR) DNA-binding protein 43 (TDP-43) is a major protein component within ubiquitin-positive inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Although TDP-43 is a nuclear DNA/RNA-binding protein, in pathological conditions, TDP-43 has been reported to redistribute to the cytoplasm where it is cleaved and forms insoluble, ubiquitinated, and phosphorylated inclusions. Here we present a cellular model in which full-length human TDP-43 or a splicing isoform (TDP-S6) that lacks the C terminus is overexpressed in a human cell line and mouse primary neurons. Whereas recombinant and endogenous TDP-43 was primarily localized in the nucleus, the shorter TDP-S6 formed highly insoluble cytoplasmic and nuclear inclusions reminiscent of disease-specific pathology. Western blot analysis of detergent-insoluble extracts showed an increase in high molecular weight immunoreactive species for TDP-S6 compared with TDP-43, consistent with ubiquitination or ubiquitin-like modifications. We used a multiplex stable isotope labeling with amino acids in cell culture approach to compare the detergent-insoluble proteome from mock-, TDP-43-, and TDP-S6-transfected cells. TDP-S6 overexpression caused a concomitant increase in both ubiquitin (Ub) and the small Ub-like modifier-2/3 (SUMO-2/3) within the insoluble proteome. Similarly, full-length TDP-43 overexpression also resulted in the elevation of SUMO-2/3. Immunofluorescence showed strong co-localization of endogenous Ub with both cytoplasmic and nuclear TDP-S6 inclusions, whereas SUMO-2/3 was co-localized mainly with the nuclear inclusions. Quantitative mass spectrometry further revealed that mixed Lys-48 and Lys-63 polyUb linkages were associated with the TDP insoluble fractions. Together our data indicate that expression of a TDP-43 splice variant lacking a C terminus recapitulates many of the cellular and biochemical features associated with disease pathology and that the interplay of ubiquitination and SUMOylation may have an important role in TDP-43 regulation.

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Genetic analysis reveals functions of atypical polyubiquitin chains.

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Genetic analysis reveals functions of atypical polyubiquitin chains.

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