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An in vivo expression system for the identification of cargo proteins of vacuolar sorting receptors in Arabidopsis culture cells

Authors

  • Jinbo Shen,

    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
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  • Pui Kit Suen,

    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
    Current affiliation:
    1. Department of Orthopaedics and Traumatology, The Chinese University of Hong Kong, Hong Kong, China
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  • Xiangfeng Wang,

    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
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  • Youshun Lin,

    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
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  • Sze Wan Lo,

    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
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  • Enrique Rojo,

    1. Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain
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  • Liwen Jiang

    Corresponding author
    1. School of Life Sciences, Centre for Cell and Developmental Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Hong Kong, China
    2. CUHK Shenzhen Research Institute, The Chinese University of Hong Kong, Shenzhen, China
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  • Upon request, all novel materials described in this publication will be made available in a timely manner for non-commercial research purposes, subject to the requisite permission from any third-party owners of all or parts of the material. Obtaining any permission will be the responsibility of the requestor.

Summary

Vacuolar sorting receptors (VSRs) are type I integral membrane family proteins that in plant cells are thought to recognize cargo proteins at the late Golgi or trans-Golgi network (TGN) for vacuolar transport via the pre-vacuolar compartment (PVC). However, little is known about VSR cargo proteins in plants. Here we developed and tested an in vivo expression system for the identification of VSR cargos which is based on the premise that the expressed N-terminus of VSRs will be secreted into the culture medium along with their corresponding cargo proteins. Indeed, transgenic Arabidopsis culture cell lines expressing VSR N-terminal binding domains (VSRNTs) were shown to secrete truncated VSRs (BP80NT, AtVSR1NT and AtVSR4NT) with attached cargo molecules into the culture medium. Putative cargo proteins were identified through mass spectrometry. Several identified cargo proteins were confirmed by localization studies and interaction analysis with VSRs. The screening strategy described here should be applicable to all VSRs and will help identify and study cargo proteins for individual VSR proteins. This method should be useful for both cargo identification and protein–protein interaction in vivo.

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