RESUMO
Mono-ubiquitinated histone H2B (H2B-Ub) is important for chromatin regulation of transcription, chromatin assembly, and also influences heterochromatin. In this review, we discuss the effects of H2B-Ub from nucleosome to higher-order chromatin structure. We then assess what is currently known of the role of H2B-Ub in heterochromatic silencing in budding and fission yeasts (S. cerevisiae and S. pombe), which have distinct silencing mechanisms. In budding yeast, the SIR complex initiates heterochromatin assembly with the aid of a H2B-Ub deubiquitinase, Ubp10. In fission yeast, the RNAi-dependent pathway initiates heterochromatin in the context of low H2B-Ub. We examine how the different silencing machineries overcome the challenge of H2B-Ub chromatin and highlight the importance of using these microorganisms to further our understanding of H2B-Ub in heterochromatic silencing pathways.
Assuntos
Heterocromatina/genética , Histonas/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Ubiquitina Tiolesterase/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Saccharomycetales/genética , Schizosaccharomyces/genética , Telômero/genética , Ubiquitina/genéticaRESUMO
Snf1, a member of the AMP-activated protein kinase family, plays a critical role in metabolic energy control in yeast cells. Snf1 activity is activated by phosphorylation of Thr-210 on the activation loop of its catalytic subunit; following activation, Snf1 regulates stress-responsive transcription factors. Here, we report that the level of Snf1 protein is dramatically decreased in a UBP8- and UBP10-deleted yeast mutant (ubp8Δ ubp10Δ), and this is independent of transcriptional regulation and proteasome-mediated degradation. Surprisingly, most Snf1-mediated functions, including glucose limitation regulation, utilization of alternative carbon sources, stress responses, and aging, are unaffected in this strain. Snf1 phosphorylation in ubp8Δ ubp10Δ cells is hyperactivated upon stress, which may compensate for the loss of the Snf1 protein and protect cells against stress and aging. Furthermore, artificial elevation of Snf1 phosphorylation (accomplished through deletion of REG1, which encodes a protein that regulates Snf1 dephosphorylation) restored Snf1 protein levels and the regulation of Snf1 activity in ubp8Δ ubp10Δ cells. Our results reveal the existence of a feedback loop that controls Snf1 protein level and its phosphorylation, which is masked by Ubp8 and Ubp10 through an unknown mechanism. We propose that this dynamic modulation of Snf1 phosphorylation and its protein level may be important for adaptation to environmental stress.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Adaptação Biológica , Retroalimentação Fisiológica , Regulação Fúngica da Expressão Gênica , Mutação , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologiaRESUMO
In the canonical view of protein function, it is generally accepted that the three-dimensional structure of a protein determines its function. However, the past decade has seen a dramatic growth in the identification of proteins with extensive intrinsically disordered regions (IDRs), which are conformationally plastic and do not appear to adopt single three-dimensional structures. One current paradigm for IDR function is that disorder enables IDRs to adopt multiple conformations, expanding the ability of a protein to interact with a wide variety of disparate proteins. The capacity for many interactions is an important feature of proteins that occupy the hubs of protein networks, in particular protein-modifying enzymes that usually have a broad spectrum of substrates. One such protein modification is ubiquitination, where ubiquitin is attached to proteins through ubiquitin ligases (E3s) and removed through deubiquitinating enzymes. Numerous proteomic studies have found that thousands of proteins are dynamically regulated by cycles of ubiquitination and deubiquitination. Thus, how these enzymes target their wide array of substrates is of considerable importance for understanding the function of the cell's diverse ubiquitination networks. Here, we characterize a yeast deubiquitinating enzyme, Ubp10, that possesses IDRs flanking its catalytic protease domain. We show that Ubp10 possesses multiple, distinct binding modules within its IDRs that are necessary and sufficient for directing protein interactions important for Ubp10's known roles in gene silencing and ribosome biogenesis. The human homolog of Ubp10, USP36, also has IDRs flanking its catalytic domain, and these IDRs similarly contain binding modules important for protein interactions. This work highlights the significant protein interaction scaffolding abilities of IDRs in the regulation of dynamic protein ubiquitination.
Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sítios de Ligação , Domínio Catalítico , Humanos , Proteínas Nucleares/química , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina Tiolesterase/químicaRESUMO
Ubiquitination controls numerous cellular processes, and its deregulation is associated with many pathologies. The Nse1 subunit in the Smc5/6 complex contains a RING domain with ubiquitin E3 ligase activity and essential functions in genome integrity. However, Nse1-dependent ubiquitin targets remain elusive. Here, we use label-free quantitative proteomics to analyze the nuclear ubiquitinome of nse1-C274A RING mutant cells. Our results show that Nse1 impacts the ubiquitination of several proteins involved in ribosome biogenesis and metabolism that, importantly, extend beyond canonical functions of Smc5/6. In addition, our analysis suggests a connection between Nse1 and RNA polymerase I (RNA Pol I) ubiquitination. Specifically, Nse1 and the Smc5/6 complex promote ubiquitination of K408 and K410 in the clamp domain of Rpa190, a modification that induces its degradation in response to blocks in transcriptional elongation. We propose that this mechanism contributes to Smc5/6-dependent segregation of the rDNA array, the locus transcribed by RNA Pol I.