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Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components.

ABSTRACT: DYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA (TorA). TorA localizes within the lumen of the nuclear envelope/endoplasmic reticulum and binds to a membrane-spanning cofactor, lamina associated polypeptide 1 (LAP1) or lumenal domain like LAP1 (LULL1), to form an ATPase; the substrate(s) of TorA remains ill-defined. Here we use budding yeast, which lack Torsins, to interrogate TorA function. We show that TorA accumulates at nuclear envelope-embedded spindle pole bodies (SPBs) in a way that requires its oligomerization and the SUN (Sad1 and UNc-84)-domain protein, Mps3. We further show that TorA physically interacts with human SUN1/2 within this system, supporting the physiological relevance of these interactions. Consistent with the idea that TorA acts on a SPB substrate, its binding to SPBs is modulated by the ATPase-stimulating activity of LAP1. TorA and TorA-?E reduce the fitness of cells expressing mps3 alleles, whereas TorA alone inhibits growth of cells lacking Pom152, a component of the nuclear pore complex. This genetic specificity is mirrored biochemically as TorA, but not TorA-?E, binds Pom152. Thus, TorA-nucleoporin interactions might be abrogated by TorA-?E, suggesting new experimental avenues to interrogate the molecular basis behind nuclear envelope herniations seen in mammalian cells lacking TorA function.

SUBMITTER: Chalfant M

PROVIDER: S-EPMC6589686 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components.

Chalfant Madeleine M Barber Karl W KW Borah Sapan S Thaller David D Lusk C Patrick CP

Molecular biology of the cell 20190109 5

DYT1 dystonia is caused by an in-frame deletion of a glutamic acid codon in the gene encoding the AAA+ ATPase TorsinA (TorA). TorA localizes within the lumen of the nuclear envelope/endoplasmic reticulum and binds to a membrane-spanning cofactor, lamina associated polypeptide 1 (LAP1) or lumenal domain like LAP1 (LULL1), to form an ATPase; the substrate(s) of TorA remains ill-defined. Here we use budding yeast, which lack Torsins, to interrogate TorA function. We show that TorA accumulates at nu ...[more]

PMID: 30625036

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Project description:BackgroundDYT1 dystonia is an autosomal dominant neurological condition caused by a mutation that removes a single glutamic acid residue (ΔE) from the torsinA (torA) AAA+ protein. TorA appears to possess a nuclear envelope (NE) localized activity that requires Lamina-Associated-Polypeptide 1 (LAP1), which is an inner nuclear membrane localized torA-binding partner. Although hypoactive, the DYT1 dystonia torA-ΔE isoform often concentrates in the NE, suggesting that torA-ΔE also interacts with an NE-localized binding partner.ResultsWe confirm that NE-localized torA-ΔE does not co-immunoprecipitate with LAP1, and find that torA-ΔE continues to concentrate in the NE of cells that lack LAP1. Instead, we find that variability in torA-ΔE localization correlates with the presence of the SUN-domain and Nesprin proteins that assemble into the LINC complex. We also find that siRNA depletion of SUN1, but not other LINC complex components, removes torA-ΔE from the NE. In contrast, the LAP1-dependent NE-accumulation of an ATP-locked torA mutant is unaffected by loss of LINC complex proteins. This SUN1 dependent torA-ΔE localization requires the torA membrane association domain, as well as a putative substrate-interaction residue, Y147, neither of which are required for torA interaction with LAP1. We also find that mutation of these motifs, or depletion of SUN1, decreases the amount of torA-WT that colocalizes with NE markers, indicating that each also underlies a normal NE-localized torA binding interaction.ConclusionsThese data suggest that the disease causing ΔE mutation promotes an association between torA and SUN1 that is distinct to the interaction between LAP1 and ATP-bound torA. This evidence for two NE-localized binding partners suggests that torA may act on multiple substrates and/or possesses regulatory co-factor partners. In addition, finding that the DYT1 mutation causes abnormal association with SUN1 implicates LINC complex dysfunction in DYT1 dystonia pathogenesis, and suggests a gain-of-function activity contributes to this dominantly inherited disease.

Dataset Information

Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components.

Publications

Expression of TorsinA in a heterologous yeast system reveals interactions with lumenal domains of LINC and nuclear pore complex components.

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