The mechanism used by mouse polyomavirus (MPyV) overcomes the crowded cytosol to reach the nucleus has not been fully elucidated. Here, we investigated the involvement of importin α/β1 mediated transport in the delivery of MPyV genomes into the nucleus. Interactions of the virus with importin β1 were studied by co-immunoprecipitation and proximity ligation assay. For infectivity and nucleus delivery assays, the virus and its capsid proteins mutated in the nuclear localization signals (NLSs) were prepared and produced. We found that at early times post infection, virions bound importin β1 in a time dependent manner with a peak of interactions at 6 h post infection. Mutation analysis revealed that only when the NLSs of both VP1 and VP2/3 were disrupted, virus did not bind efficiently to importin β1 and its infectivity remarkably decreased (by 80%). Nuclear targeting of capsid proteins was improved when VP1 and VP2 were co-expressed. VP1 and VP2 were effectively delivered into the nucleus, even when one of the NLS, either VP1 or VP2, was disrupted. Altogether, our results showed that MPyV virions can use VP1 and/or VP2/VP3 NLSs in concert or individually to bind importins to deliver their genomes into the cell nucleus.

• MeSH
• Biological Transport MeSH
• Cell Nucleus MeSH
• Cell Line MeSH
• DNA, Viral metabolism   MeSH
• Fluorescent Antibody Technique MeSH
• Nuclear Localization Signals genetics   MeSH
• Karyopherins metabolism   MeSH
• Mutation MeSH
• Mice MeSH
• Polyomavirus Infections metabolism   virology   MeSH
• Polyomavirus physiology   ultrastructure   MeSH
• Virus Assembly MeSH
• Amino Acid Substitution MeSH
• Protein Binding MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Although telomerase (EC 2.7.7.49) is important for genome stability and totipotency of plant cells, the principles of its regulation are not well understood. Therefore, we studied subcellular localization and function of the full-length and truncated variants of the catalytic subunit of Arabidopsis thaliana telomerase, AtTERT, in planta. Our results show that multiple sites in AtTERT may serve as nuclear localization signals, as all the studied individual domains of the AtTERT were targeted to the nucleus and/or the nucleolus. Although the introduced genomic or cDNA AtTERT transgenes display expression at transcript and protein levels, they are not able to fully complement the lack of telomerase functions in tert -/- mutants. The failure to reconstitute telomerase function in planta suggests a more complex telomerase regulation in plant cells than would be expected based on results of similar experiments in mammalian model systems.

• MeSH
• Arabidopsis enzymology   genetics   MeSH
• Cell Nucleolus enzymology   genetics   MeSH
• Cell Nucleus enzymology   genetics   MeSH
• Plants, Genetically Modified MeSH
• Nuclear Localization Signals genetics   MeSH
• Catalytic Domain genetics   MeSH
• Plant Leaves genetics   MeSH
• Arabidopsis Proteins genetics   metabolism   MeSH
• Protein Biosynthesis MeSH
• Gene Expression Regulation, Plant MeSH
• RNA Splicing MeSH
• Nicotiana genetics   MeSH
• Telomerase chemistry   genetics   metabolism   MeSH
• Protein Structure, Tertiary MeSH
• Structure-Activity Relationship MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The protein Isw1 of Saccharomyces cerevisiae is an imitation-switch chromatin-remodeling factor. We studied the mechanisms of its nuclear import and found that the nuclear localization signal (NLS) mediating the transport of Isw1 into the nucleus is located at the end of the C-terminus of the protein (aa1079-1105). We show that it is an atypical bipartite signal with an unconventional linker of 19 aa (KRIR X(19) KKAK) and the only nuclear targeting signal within the Isw1 molecule. The efficiency of Isw1 nuclear import was found to be modulated by changes to the amino acid composition in the vicinity of the KRIR motif, but not by the linker length. Live-cell imaging of various karyopherin mutants and in vitro binding assays of Isw1NLS to importin-α revealed that the nuclear translocation of Isw1 is mediated by the classical import pathway. Analogous motifs to Isw1NLS are highly conserved in Isw1 homologues of other yeast species, and putative bipartite cNLS were identified in silico at the end of the C-termini of imitation switch (ISWI) proteins from higher eukaryotes. We suggest that the C-termini of the ISWI family proteins play an important role in their nuclear import.

• MeSH
• Adenosine Triphosphatases chemistry   genetics   metabolism   MeSH
• Active Transport, Cell Nucleus genetics   MeSH
• Amino Acid Motifs MeSH
• Cell Nucleus metabolism   MeSH
• DNA-Binding Proteins chemistry   genetics   metabolism   MeSH
• Nuclear Localization Signals * genetics   MeSH
• Mutation MeSH
• Nucleocytoplasmic Transport Proteins genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH