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bioRxiv · 10.1101/2025.04.28.651124

Macromolecular Crowding-Affected Mobility of Bcnt/Cfdp1, a Disordered Component of the Srcap Complex, from the Nucleus to Cytosol in Subcellular Fractionation

Iwashita, S.·Suzuki, T.·Nishimura, K.·Onishi, A.·Tsukuda, K.·Kakihara, Y.·Kiriyama, Y.·Ohoka, Y.·Nakashima, K.
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Abstract

Metazoan Bucentaur/Craniofacial Development Protein 1 (Bcnt/Cfdp1) is crucial for embryonic development, and its yeast homolog protein, SWC5, functions as part of the SWR1 complex in histone exchange reactions. Bcnt/Cfdp1, an intrinsically disordered protein (IDP) subject to various post-translational modifications, contains a putative nuclear localization sequence. Nevertheless, its subcellular localization remains ambiguous. We investigated its intracellular trafficking using diverse approaches. In C2C12 myoblasts, fluorescent protein-tagged Bcnt/Cfdp1 was predominantly localized within the nucleus, preferentially in regions of low DAPI density in myoblasts, and the localization persisted in differentiated multinucleated myotubes. Conversely, detergent-based biochemical fractionation of C2C12 and HEK293T cells indicated a substantial presence of Bcnt/Cfdp1 in cytoplasmic fractions. Quantitative proteomic analysis of fractionated proteins revealed that six IDPs, including a PEST-containing nuclear protein, demonstrated similar distribution patterns to Bcnt/Cfdp1; despite being annotated as nuclear proteins, they were detected in cytoplasmic fractions. Direct treatment of both cell lines with digitonin enabled the collection of released proteins in three steps to accurately isolate cytoplasmic proteins. Bcnt/Cfdp1 exhibited distinct extraction patterns compared to cytoplasmic soluble protein extracts in Ficoll/sucrose solution, while no differences were noted when cells underwent digitonin treatment in an isotonic solution. Quantitative proteomic analysis of these extracts highlighted the mobility or integrity of each protein within the cytoplasm. Consequently, although Bcnt/Cfdp1 is essential within the nucleus, our findings suggest its additional presence in the cytoplasm. We hypothesize that the elastic properties of Bcnt/Cfdp1 facilitate its translocation from the nucleus during subcellular fractionation, which is affected by macromolecular crowding.

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Iwashita, S., Suzuki, T., Nishimura, K., Onishi, A., Tsukuda, K., Kakihara, Y., Kiriyama, Y., Ohoka, Y., Nakashima, K.. 2025-04-29. Macromolecular Crowding-Affected Mobility of Bcnt/Cfdp1, a Disordered Component of the Srcap Complex, from the Nucleus to Cytosol in Subcellular Fractionation. https://doi.org/10.1101/2025.04.28.651124

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