ABSTRACT
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.
Subject(s)
ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases , Methyltransferases , Animals , Humans , Adaptor Proteins, Signal Transducing/metabolism , Adenosine Triphosphatases/metabolism , ATPases Associated with Diverse Cellular Activities/metabolism , Lysine/metabolism , Methylation , Protein Binding , Protein Processing, Post-Translational , Transcription Factors/metabolism , Valosin Containing Protein/metabolism , Methyltransferases/metabolismSubject(s)
AAA Proteins/genetics , ATPases Associated with Diverse Cellular Activities/ultrastructure , Endosomal Sorting Complexes Required for Transport/ultrastructure , Enzyme Inhibitors/chemistry , Protein Conformation/drug effects , AAA Proteins/antagonists & inhibitors , AAA Proteins/ultrastructure , ATPases Associated with Diverse Cellular Activities/antagonists & inhibitors , Adenosine Triphosphate/genetics , Crystallography, X-Ray , Endosomal Sorting Complexes Required for Transport/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Humans , HydrolysisABSTRACT
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.
Subject(s)
ATPases Associated with Diverse Cellular Activities/chemistry , Adenosine Triphosphatases/chemistry , Arabidopsis Proteins/chemistry , Arabidopsis/genetics , Carrier Proteins/chemistry , Cell Cycle Proteins/chemistry , Coenzymes/chemistry , Intracellular Signaling Peptides and Proteins/chemistry , Nuclear Proteins/chemistry , ATPases Associated with Diverse Cellular Activities/genetics , ATPases Associated with Diverse Cellular Activities/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Amino Acid Motifs , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Binding Sites , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cloning, Molecular , Coenzymes/genetics , Coenzymes/metabolism , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Models, Molecular , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Protein Stability , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structural Homology, Protein , Substrate SpecificityABSTRACT
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.
