RESUMO
Ubiquitin has seven lysines, all of which are used to generate polyubiquitin chains in the yeast Saccharomyces cerevisiae. While the biology associated with chains formed through lysines 48 and 63 is well studied, other chain types are more poorly characterized. We outline a methodology for using synthetic genetic analysis to examine ubiquitin mutants. Ubiquitin is encoded by four loci, necessitating several alterations to standard protocols, including the use of the SK1 strain background, which sporulates with very high efficiency. The methods described here could be used to examine other ubiquitin mutants, including those that do not support viability.
Assuntos
Poliubiquitina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismo , Lisina/genética , Lisina/metabolismo , Mutação , Poliubiquitina/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/genética , Arginina/metabolismo , Regulação Fúngica da Expressão Gênica , Lisina/metabolismo , Poliubiquitina/genética , Saccharomyces cerevisiae/genética , Ubiquitina/genética , Substituição de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Arginina/genética , Transporte Biológico , Ciclo Celular/genética , Engenharia Genética , Lisina/genética , Análise em Microsséries , Poliubiquitina/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
CDC20 and CDH1 are well-established substrate receptors for the Anaphase Promoting Complex/Cyclosome (APC/C). In this issue of Molecular Cell, Lee et al. (2015) show that these adaptors can also target cell cycle proteins for destruction through a second ubiquitin ligase, Parkin.
Assuntos
Caderinas/metabolismo , Proteínas Cdc20/metabolismo , Instabilidade Genômica , Mitose , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , HumanosRESUMO
The SCF ubiquitin ligase associates with substrates through its F-box protein adaptor. Substrates are typically recognized through a defined phosphodegron. Here, we characterize the interaction of the F-box protein Saf1 with Prb1, one of its vacuolar protease substrates. We show that Saf1 binds the mature protein but ubiquitinates only the zymogen precursor. The ubiquitinated lysine was found to be in a peptide eliminated from the mature protein. Mutations that eliminate the catalytic activity of Prb1, or the related substrate Prc1, block Saf1 targeting of the zymogen precursor. Our data suggest that Saf1 does not require a conventional degron as do other F-box proteins but instead recognizes the catalytic site itself.
