Aaa Atpase
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2021 ◽  
Vol 12 ◽  
Heike Wolff ◽  
Marc Jakoby ◽  
Lisa Stephan ◽  
Eva Koebke ◽  
Martin Hülskamp

The Arabidopsis AAA ATPase SKD1 is essential for ESCRT-dependent endosomal sorting by mediating the disassembly of the ESCRTIII complex in an ATP-dependent manner. In this study, we show that SKD1 localizes to messenger ribonucleoprotein complexes upon heat stress. Consistent with this, the interactome of SKD1 revealed differential interactions under normal and stress conditions and included membrane transport proteins as well as proteins associated with RNA metabolism. Localization studies with selected interactome proteins revealed that not only RNA associated proteins but also several ESCRTIII and membrane trafficking proteins were recruited to messenger ribonucleoprotein granules after heat stress.

2021 ◽  
Vol 220 (8) ◽  
Chun-Che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

2021 ◽  
Vol 12 (1) ◽  
Michael Prattes ◽  
Irina Grishkovskaya ◽  
Victor-Valentin Hodirnau ◽  
Ingrid Rössler ◽  
Isabella Klein ◽  

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

eLife ◽  
2021 ◽  
Vol 10 ◽  
Sudeep Banjade ◽  
Yousuf H Shah ◽  
Shaogeng Tang ◽  
Scott D Emr

ESCRT-III polymerization is required for all endosomal sorting complex required for transport (ESCRT)-dependent events in the cell. However, the relative contributions of the eight ESCRT-III subunits differ between each process. The minimal features of ESCRT-III proteins necessary for function and the role for the multiple ESCRT-III subunits remain unclear. To identify essential features of ESCRT-III subunits, we previously studied the polymerization mechanisms of two ESCRT-III subunits Snf7 and Vps24, identifying the association of the helix-4 region of Snf7 with the helix-1 region of Vps24 (Banjade et al., 2019a). Here, we find that mutations in the helix-1 region of another ESCRT-III subunit Vps2 can functionally replace Vps24 in Saccharomyces cerevisiae. Engineering and genetic selections revealed the required features of both subunits. Our data allow us to propose three minimal features required for ESCRT-III function – spiral formation, lateral association of the spirals through heteropolymerization, and binding to the AAA + ATPase Vps4 for dynamic remodeling.

2021 ◽  
Drisya Vijayakumari ◽  
Janina Müller ◽  
Silke Hauf

SummaryCdc48 (p97/VCP) is a AAA-ATPase that can extract ubiquitinated proteins from their binding partners and channel substrates to the proteasome. A fission yeast cdc48 mutant (cdc48-353) shows low levels of the cohesin protease, separase, and pronounced chromosome segregation defects in mitosis. Separase initiates chromosome segregation when its binding partner securin is ubiquitinated and degraded. The low separase levels in the cdc48-353 mutant have been attributed to a failure to extract ubiquitinated securin from separase resulting in co-degradation of separase along with securin. If true, this establishes Cdc48 as a key regulator of mitosis. In contrast, we show here that low separase levels in the cdc48-353 mutant are independent of mitosis. Moreover, we find no evidence of enhanced separase degradation in the mutant. Instead, we suggest that the cdc48-353 mutant uncovers specific requirements for separase translation. Our results highlight a need to better understand how this key mitotic enzyme is synthesized.

2021 ◽  
Vol 13 (587) ◽  
pp. eabg1168
Blandine Roux ◽  
Camille Vaganay ◽  
Jesse D. Vargas ◽  
Gabriela Alexe ◽  
Chaima Benaksas ◽  

The development and survival of cancer cells require adaptive mechanisms to stress. Such adaptations can confer intrinsic vulnerabilities, enabling the selective targeting of cancer cells. Through a pooled in vivo short hairpin RNA (shRNA) screen, we identified the adenosine triphosphatase associated with diverse cellular activities (AAA-ATPase) valosin-containing protein (VCP) as a top stress-related vulnerability in acute myeloid leukemia (AML). We established that AML was the most responsive disease to chemical inhibition of VCP across a panel of 16 cancer types. The sensitivity to VCP inhibition of human AML cell lines, primary patient samples, and syngeneic and xenograft mouse models of AML was validated using VCP-directed shRNAs, overexpression of a dominant-negative VCP mutant, and chemical inhibition. By combining mass spectrometry–based analysis of the VCP interactome and phospho-signaling studies, we determined that VCP is important for ataxia telangiectasia mutated (ATM) kinase activation and subsequent DNA repair through homologous recombination in AML. A second-generation VCP inhibitor, CB-5339, was then developed and characterized. Efficacy and safety of CB-5339 were validated in multiple AML models, including syngeneic and patient-derived xenograft murine models. We further demonstrated that combining DNA-damaging agents, such as anthracyclines, with CB-5339 treatment synergizes to impair leukemic growth in an MLL-AF9–driven AML murine model. These studies support the clinical testing of CB-5339 as a single agent or in combination with standard-of-care DNA-damaging chemotherapy for the treatment of AML.

2021 ◽  
Richard Cardoso da Silva ◽  
Gerben Vader

AbstractThe generally conserved AAA+ ATPase Pch2/TRIP13 is involved in diverse aspects of meiosis, such as prophase checkpoint function, DNA break regulation, and meiotic recombination. The controlled recruitment of Pch2 to meiotic chromosomes allows it to use its ATPase activity to influence HORMA protein-dependent signaling. Because of the connection between Pch2 chromosomal recruitment and its functional roles in meiosis, it is important to reveal the molecular details that govern Pch2 localization. Here, we review the current understanding of the different factors that control the recruitment of Pch2 to meiotic chromosomes, with a focus on research performed in budding yeast. During meiosis in this organism, Pch2 is enriched within the nucleolus, where it likely associates with the specialized chromatin of the ribosomal (r)DNA. Pch2 is also found on non-rDNA euchromatin, where its recruitment is contingent on Zip1, a component of the synaptonemal complex (SC) that assembles between homologous chromosomes. We discuss recent findings connecting the recruitment of Pch2 with its association with the Origin Recognition Complex (ORC) and reliance on RNA Polymerase II-dependent transcription. In total, we provide a comprehensive overview of the pathways that control the chromosomal association of an important meiotic regulator.

2021 ◽  
pp. jcs.254201
Sirisha Mukkavalli ◽  
Jacob Aaron Klickstein ◽  
Betty Ortiz ◽  
Peter Juo ◽  
Malavika Raman

The recognition and disposal of misfolded proteins is essential for the maintenance of cellular homeostasis. Perturbations in the pathways that promote degradation of aberrant proteins contribute to a variety of protein aggregation disorders broadly termed proteinopathies. The p97 AAA-ATPase in combination with adaptor proteins functions to identify ubiquitylated proteins and target them for degradation by the proteasome or autophagy. Mutations in p97 cause multi-system proteinopathies; however, the precise defects underlying these disorders are unclear. Here, we systematically investigate the role of p97 and its adaptors in the process of formation of aggresomes, membrane-less structures containing ubiquitylated proteins that arise upon proteasome inhibition. We demonstrate that p97 mediates aggresome formation and clearance and identify a novel role for the adaptor UBXN1 in the process of aggresome formation. UBXN1 is recruited to aggresomes and UBXN1 knockout cells are unable to form aggresomes. Loss of p97-UBXN1 results in increased Huntingtin polyQ inclusion bodies both in mammalian cells as well as in a C.elegans model of Huntington's Disease. Together our work identifies evolutionarily conserved roles for p97-UBXN1 in the disposal of protein aggregates.

2021 ◽  
Ritam Das ◽  
Urmi Bajpai

AbstractMycobacteriophages are viruses of Mycobacterium spp. with promising diagnostic and therapeutic potential. Phage genome exploration and characterization of their proteomes are essential to gain a better understanding of their role in phage biology. So far, about 2014 mycobacteriophages have been genomically defined and 1563 phage protein families (phamilies) are identified. However, the function of only a fraction (about 15%) is known and a majority of ORFs in phage genomes are hypothetical proteins. In this study, from the annotated genome of a F1 cluster mycobacteriophage SimranZ1, a putative AAA ATPase (Gp65, Pham 9410) is characterized as a DNA-dependent ATPase. Sequence-based functional annotation predicted Gp65 to belong to the P-loop NTPase superfamily, having AAA_24 and RecA/RadA domains which are known to be involved in ATP-dependent DNA repair/maintenance mechanism. On molecular docking, Gly21 and Ser23 of Gp65 showed specific binding with ATP. Using a microtiter plate assay, ATPase activity of Gp65 was experimentally verified which was found to increase in the presence of dsDNA. Gel electrophoresis under non-denaturing condition showed the oligomeric states of Gp65 and Transmission Electron Microscopy revealed it to exist as a hexamer having a prominent central pore with a diameter of 1.9 nm. In summary, functional characterization of Gp65 as a DNA dependent AAA ATPase indicates its role in DNA repair/maintenance mechanism in mycobacteriophages.

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