Drosophila proteins involved in metabolism of uracil‐DNA possess different types of nuclear localization signals

Abstract

Adequate transport of large proteins that function in the nucleus is indispensable for cognate molecular events within this organelle. Selective protein import into the nucleus requires nuclear localization signals (NLS) that are recognized by importin receptors in the cytoplasm. Here we investigated the sequence requirements for nuclear targeting of Drosophila proteins involved in the metabolism of uracil‐substituted DNA: the recently identified uracil‐DNA degrading factor, dUTPase, and the two uracil‐DNA glycosylases present in Drosophila. For the uracil‐DNA degrading factor, NLS prediction identified two putative NLS sequences [PEKRKQE(320–326) and PKRKKKR(347–353)]. Truncation and site‐directed mutagenesis using YFP reporter constructs showed that only one of these basic stretches is critically required for efficient nuclear localization in insect cells. This segment corresponds to the well‐known prototypic NLS of SV40 T‐antigen. An almost identical NLS segment is also present in the Drosophila thymine‐DNA glycosylase, but no NLS elements were predicted in the single‐strand‐specific monofunctional uracil‐DNA glycosylase homolog protein. This latter protein has a molecular mass of 31 kDa, which may allow NLS‐independent transport. For Drosophila dUTPase, two isoforms with distinct features regarding molecular mass and subcellular distribution were recently described. In this study, we characterized the basic PAAKKMKID(10–18) segment of dUTPase, which has been predicted to be a putative NLS by in silico analysis. Deletion studies, using YFP reporter constructs expressed in insect cells, revealed the importance of the PAA(10–12) tripeptide and the ID(17–18) dipeptide, as well as the role of the PAAK(10–13) segment in nuclear localization of dUTPase. We constructed a structural model that shows the molecular basis of such recognition in three dimensions.

Analysis of Sub-Lethal Toxicity of Perfluorooctane Sulfonate (PFOS) to Daphnia magna Using 1H Nuclear Magnetic Resonance-Based Metabolomics

“…Uracil plays various roles in metabolic activity, one of which is the regulation of coenzymes including the role of converting glucose to galactose [55,56]. The observed increase in uracil, with increasing PFOS exposure, suggests an attempt at enzyme regulation in response to the deregulation being caused by PFOS toxicity.…”

Calpain-Catalyzed Proteolysis of Human dUTPase Specifically Removes the Nuclear Localization Signal Peptide

“…The NLS segment shows conserved character with respect to e.g. Drosophila dUTPase where its lack was also shown to lead to exclusion of dUTPase from the cell nucleus [26]. Without this signal dUTPase is unable to enter the nucleus and may not function correctly.…”

“…50 kDa molecular mass of these proteins that prevents passive transport. Nuclear availability of the enzyme seems to be generally required for eukaryotic cells, attested to by the usual presence of a nuclear localization signal at the N-terminus [24], [26], [28]. Interestingly, for dUTPase from Singapore grouper iridovirus (a fish virus), a nuclear export signal was also described, indicating that trafficking of the host and the pathogen enzyme may be different [29].…”

Association of RNA with the uracil‐DNA‐degrading factor has major conformational effects and is potentially involved in protein folding

“…1A, 1B, 2, 3, and 4: conserved motifs of UDE. NLS: nuclear localization signal at the end of the sequence [17]. Black and grey arrows indicate the preferred cleavage sites of ArgC and AspN, respectively.…”

“…RNA binding to UDE may have different physiological roles: (a) RNA may stabilize the conformation of UDE protein; (b) RNA may inhibit UDE activity; (c) RNA may influence the cellular localization of the protein; and (d) UDE may have an as yet unidentified role in either stabilization, localization or regulation of RNAs. Nuclear localization of UDE was suggested by both nuclear localization signal (NLS) analysis of green fluorescent protein fusion constructs [17], and immunostaining of S2 cells (unpublished). However, detailed characterization of UDE localization in tissues awaits further studies.…”