RESUMEN
p97 is an essential AAA+ ATPase that extracts and unfolds substrate proteins from membranes and protein complexes. Through its mode of action, p97 contributes to various cellular processes, such as membrane fusion, ER-associated protein degradation, DNA repair, and many others. Diverse p97 functions and protein interactions are regulated by a large number of adaptor proteins. Alveolar soft part sarcoma locus (ASPL) is a unique adaptor protein that regulates p97 by disassembling functional p97 hexamers to smaller entities. An alternative mechanism to regulate the activity and interactions of p97 is by posttranslational modifications (PTMs). Although more than 140 PTMs have been identified in p97, only a handful of those have been described in detail. Here we present structural and biochemical data to explain how the p97-remodeling adaptor protein ASPL enables the metastasis promoting methyltransferase METTL21D to bind and trimethylate p97 at a single lysine side chain, which is deeply buried inside functional p97 hexamers. The crystal structure of a heterotrimeric p97:ASPL:METTL21D complex in the presence of cofactors ATP and S-adenosyl homocysteine reveals how structural remodeling by ASPL exposes the crucial lysine residue of p97 to facilitate its trimethylation by METTL21D. The structure also uncovers a role of the second region of homology (SRH) present in the first ATPase domain of p97 in binding of a modifying enzyme to the AAA+ ATPase. Investigation of this interaction in the human, fish, and plant reveals fine details on the mechanism and significance of p97 trimethylation by METTL21D across different organisms.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas , Metiltransferasas , Animales , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Lisina/metabolismo , Metilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Factores de Transcripción/metabolismo , Proteína que Contiene Valosina/metabolismo , Metiltransferasas/metabolismoAsunto(s)
Proteínas AAA/genética , ATPasas Asociadas con Actividades Celulares Diversas/ultraestructura , Complejos de Clasificación Endosomal Requeridos para el Transporte/ultraestructura , Inhibidores Enzimáticos/química , Conformación Proteica/efectos de los fármacos , Proteínas AAA/antagonistas & inhibidores , Proteínas AAA/ultraestructura , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , Adenosina Trifosfato/genética , Cristalografía por Rayos X , Complejos de Clasificación Endosomal Requeridos para el Transporte/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Humanos , HidrólisisRESUMEN
The hexameric ring structure of the type II AAA+ ATPases is considered as stable and permanent. Recently, the UBX domain-containing cofactors Arabidopsis thaliana PUX1 and human alveolar soft part sarcoma locus (ASPL) were reported to bind and disassemble the cognate AAA+ ATPases AtCDC48 and human p97. Here, we present two crystal structures related to these complexes: a truncated AtCDC48 (AtCDC48-ND1) and a hybrid complex containing human p97-ND1 and the UBX domain of plant PUX1 (p97-ND1:PUX1-UBX). These structures reveal close similarity between the human and plant AAA+ ATPases, but also highlight differences between disassembling and non-disassembling AAA+ ATPase cofactors. Based on an AtCDC48 disassembly assay with PUX1 and known crystal structures of the p97-bound human cofactor ASPL, we propose a general ATPase disassembly model. Thus, our structural and biophysical investigations provide detailed insight into the mechanism of AAA+ ATPase disassembly by UBX domain cofactors and suggest a general mode of regulating the cellular activity of these molecular machines.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/química , Adenosina Trifosfatasas/química , Proteínas de Arabidopsis/química , Arabidopsis/genética , Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Coenzimas/química , Péptidos y Proteínas de Señalización Intracelular/química , Proteínas Nucleares/química , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Secuencias de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Coenzimas/genética , Coenzimas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Especificidad por SustratoRESUMEN
This paper analyzes the impact of genes and proportional contribution of parental genotypes on the inheritance of root yield and sugar content in diploid hybrids of sugar beet. The survey included two diploid male-sterile monogerm lines and three single (SC) male-sterile hybrids as maternal components, while three multigerm diploids were used as pollinators. The partitioning of genotypic variance into additive and dominant components was performed by half sibling (HS) and full sibling (FS) covariance. The proportional contribution of individual components of crossbreeding (lines, testers, and interactions) was exhibited in the expression of certain characteristics of F1 generation. Genotypic variance components showed a significant effect of nonadditive gene action (dominance) in the inheritance of root yield and sugar content, while the additive effect of genes was less significant. Maternal components had a greater proportional contribution to root yield, while lines, pollinators, and their interactions had an equal contribution to sugar content.