Abstract
BackgroundAnkyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle1 is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm. It contains a GIY-YIG-type nuclease domain, but its function is unknown. The mammalian genome encodes only one other GIY-YIG domain protein, termed Slx1. Slx1 has been described as a resolvase that processes Holliday junctions during homologous recombination-mediated DNA double strand break repair. Resolvase activity is regulated in a spatial and temporal manner during the cell cycle. We hypothesized that Ankle1 may have a similar function and its nucleo-cytoplasmic shuttling may contribute to the regulation of Ankle1 activity. Hence, we aimed at identifying the domains mediating Ankle1 shuttling and investigating whether cellular localization is affected during DNA damage response.ResultsSequence analysis predicts the presence of two canonical nuclear import and export signals in Ankle1. Immunofluorescence microscopy of cells expressing wild-type and various mutated Ankle1-fusion proteins revealed a C-terminally located classical monopartite nuclear localization signal and a centrally located CRM1-dependent nuclear export signal that mediate nucleo-cytoplasmic shuttling of Ankle1. These sequences are also functional in heterologous proteins. The predominant localization of Ankle1 in the cytoplasm, however, does not change upon induction of several DNA damage response pathways throughout the cell cycle.ConclusionsWe identified the domains mediating nuclear import and export of Ankle1. Ankle1’s cellular localization was not affected following DNA damage.Electronic supplementary materialThe online version of this article (doi:10.1186/s12860-016-0102-z) contains supplementary material, which is available to authorized users.
Highlights
Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif
Nucleocytoplasmic shuttling of Ankle1 is mediated by active transport mechanisms In a previous study, we showed that Ankle1 is primarily localized in the cytoplasm in HeLa and B-cell derived RAMOS cell lines, but it accumulated in the nucleus upon inhibition of nuclear export by leptomycin [22, 44]
We wanted to identify the domains in Ankle1 that mediate its shuttling, and test, whether nucleocytoplasmic shuttling of Ankle1 and its cellular localization change upon DNA damage or during the cell cycle
Summary
Ankyrin repeats and LEM domain containing protein 1 (Ankle1) belongs to the LEM protein family, whose members share a chromatin-interacting LEM motif. Unlike most other LEM proteins, Ankle is not an integral protein of the inner nuclear membrane but shuttles between the nucleus and the cytoplasm It contains a GIY-YIG-type nuclease domain, but its function is unknown. The LAP2-emerin-MAN1 (LEM) protein family comprises a group of inner nuclear membrane and nucleoplasmic proteins [1, 2] with important functions in various cellular processes, including nuclear envelope architecture [3], DNA replication [4], cell cycle control [5], chromatin organization [6, 7] and the regulation of gene expression. Ankle has several unique features among the LEM protein family members It lacks a transmembrane domain and shuttles between the cytoplasm and the nucleoplasm, and it contains a C-terminal GIY-YIG-type endonuclease domain [22, 24]. Ankle functions may be highly redundant, as Ankle knockout mice and cells are normal and did not show an impaired DNA damage response [29]
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