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Poster

Session: Session 1 Presenting Authors at Posters (Odds)

P-255 - Proximity labeling-based proteomics defines endoplasmic reticulum subdomain localization of methyltransferase domain of potexvirus replicase and identifies candidate host factors for viral replication

Monday, July 14, 2025
13:30 - 15:15  
Location: Confex Hall 3
Plant-virus Interactions (incl. Viromes, Evolution & Vectors)

    Presenting Author(s)

  • YC

    Yi-Chen Chang, PhD

    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Tokyo, Japan

    • Author(s)

  • ST

    Sae TAKATSUKA

    Institute of Agriculture, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

  • NS

    Nobumitsu SASAKI

    Professor
    Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

  • YI

    Yoshiyuki ITOH

    Professor
    Smart-Core Facility Promotion Organization, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

  • MH

    Miki HISADA

    Dr.
    Smart-Core Facility Promotion Organization, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

  • TA

    Tsutomu Arie

    Professor
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan
    fuchu, Tokyo, Japan

  • KK

    Ken KOMATSU

    Professor
    United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan
    Fuchu, Tokyo, Japan

The endoplasmic reticulum (ER) is employed by many (+)RNA viruses for their replication through the formation of a virus replication complex (VRC). However, the intricate interaction of ER with other subcellular organelles renders ER proteome challenging to comprehend, which is an obstacle for elucidating the unique protein composition of the VRC. In order to capture the ER proteome, we have developed a proximity labeling (PL) method using ER membrane- and lumen-targeted TurboID-fused green fluorescent protein in Nicotiana benthamiana. Using these data as a control, we proceeded to identify the significantly captured proteins proximal to the TurboID-fused methyltransferase domain (MET-TurboID) of a replicase of plantago asiatica mosaic virus, which possesses a membrane-associated capacity important for virus replication. Different predicted subcellular localizations of captured proteins obtained via streptavidin bead pulldown and mass spectrometry revealed the efficacy of this PL system in distinguishing the proximal proteome of the ER membrane from that of the ER lumen. Meanwhile, the MET-TurboID labeled much more specific host factors, such as calcium-sensing receptor and remorin, which may be involved in virus replication and reflect a potential VRC content. Moreover, combined with the findings from confocal microscopy and protein fractionation, the MET-specific proteome appeared to localize to a unique ER subdomain possibly associated with chloroplasts and mitochondria.