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Hiraoka, Y.

Publications and source records attributed to Hiraoka, Y..

3 recordsLinked to original sources
bioRxiv2017

Asymmetrical localization of components of the Nup107-160 subcomplex within the nuclear pore complex in fission yeast

The nuclear pore complex (NPC) forms a gateway for nucleocytoplasmic transport. The outer ring protein complex of the NPC (the Nup107-160 subcomplex in humans) is a key component for building the NPC. Nup107-160 subcomplexes are believed to be symmetrically localized on the nuclear and cytoplasmic sides of the NPC. However, in S. pombe immunoelectron and fluorescence microscopic analyses revealed that the homologous components of the human Nup107-160 subcomplex had an asymmetrical localization: constituent proteins spNup132 and spNup107 were present only on the nuclear side (designated the spNup132 subcomplex), while spNup131, spNup120, spNup85, spNup96, spNup37, spEly5 and spSeh1 were localized only on the cytoplasmic side (designated the spNup120 subcomplex), suggesting the complex was split into two pieces at the interface between spNup96 and spNup107. This contrasts with the symmetrical localization reported in other organisms. Fusion of spNup96 (cytoplasmic localization) with spNup107 (nuclear localization) caused cytoplasmic relocalization of spNup107. In this strain, half of the spNup132 proteins, which interact with spNup107, changed their localization to the cytoplasmic side of the NPC, leading to defects in mitotic and meiotic progression similar to an spNup132 deletion strain. These observations suggest the asymmetrical localization of the outer ring spNup132 and spNup120 subcomplexes of the NPC is necessary for normal cell cycle progression in fission yeast. Author summaryThe nuclear pore complexes (NPCs) form gateways to transport intracellular molecules between the nucleus and the cytoplasm across the nuclear envelope. The Nup107-160 subcomplex, that forms nuclear and cytoplasmic outer rings, is a key complex responsible for building the NPC by symmetrical localization on the nuclear and cytoplasmic sides of the nuclear pore. This structural characteristic was found in various organisms including humans and budding yeasts, and therefore believed to be common among "all" eukaryotes. However, in this paper, we revealed an asymmetrical localization of the homologous components of the human Nup107-160 subcomplex in fission yeast by immunoelectron and fluorescence microscopic analyses: in this organism, the Nup107-160 subcomplex is split into two pieces, and each of the split pieces is differentially distributed to the nuclear and cytoplasmic side of the NPC: one piece is only in the nuclear side while the other piece is only in the cytoplasmic side. This contrasts with the symmetrical localization reported in other organisms. In addition, we confirmed that the asymmetrical configuration of the outer ring structure is necessary for cell cycle progression in fission yeast. This study provides notions of diverse structures and functions of NPCs evolved in eukaryotes.

cell biology↗
bioRxiv2017

Exportin Crm1 is repurposed as a docking protein to generate microtubule organizing centers at the nuclear pore

Non-centrosomal microtubule organizing centers (MTOCs) are important for microtubule organization in many cell types. In fission yeast Schizosaccharomyces pombe, the protein Mto1, together with partner protein Mto2 (Mto1/2 complex), recruits the {gamma}-tubulin complex to multiple non-centrosomal MTOCs, including the nuclear envelope (NE). Here, we develop a comparative-interactome mass spectrometry approach to determine how Mto1 localizes to the NE. Surprisingly, we find that Mto1, a constitutively cytoplasmic protein, docks at nuclear pore complexes (NPCs), via interaction with exportin Crm1 and cytoplasmic FG-nucleoporin Nup146. Although Mto1 is not a nuclear export cargo, it binds Crm1 via a nuclear export signal-like sequence, and docking requires both Ran in the GTP-bound state and Nup146 FG repeats. In addition to determining the mechanism of MTOC formation at the NE, our results reveal a novel role for Crm1 and the nuclear export machinery in the stable docking of a cytoplasmic protein complex at NPCs.

cell biology↗
bioRxiv2017

Compositionally distinct nuclear pore complexes of functionally distinct dimorphic nuclei in ciliate Tetrahymena

SUMMARY STATEMENTOur study demonstrates compositional and structural differences of the nuclear pore complex between the functionally differentiated macronucleus and micronucleus within a single cytoplasm of ciliated protozoa.\n\nABSTRACTThe nuclear pore complex (NPC), a gateway for nucleocytoplasmic trafficking, is composed of about 30 different proteins called nucleoporins. It remains unknown whether the NPCs within a species are homogeneous or vary depending on the cell type, or physiological condition. Here, we present evidence for compositionally distinct NPCs that form within a single cell in a binucleated ciliate. In Tetrahymena thermophila, each cell contains both a transcriptionally-active macronucleus (MAC) and a germline micronucleus (MIC). By combining in silico analysis, mass spectrometry analysis for immuno-isolated proteins, and subcellular localization analysis of GFP fused proteins, we identified numerous novel components of MAC and MIC NPCs. Core members of the Nup107-160 scaffold complex were enriched in MIC NPCs. Strikingly, two paralogs of Nup214 and of Nup153 localized exclusively to either MAC or MIC NPCs. Furthermore, the transmembrane components Pom121 and Pom82 localize exclusively to MAC and MIC NPCs, respectively. Our results argue that functional nuclear dimorphism in ciliates is likely to depend on compositional and structural specificity of NPCs.

cell biology↗