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Quantification of Protein Exit at the Trans-Golgi Network

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Golgi

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2557))

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Abstract

With one-third of all newly synthesized proteins entering the secretory pathway, correct protein sorting is essential for cellular homeostasis. In the last three decades, researchers have developed numerous biochemical, genetic, and cell biological approaches to study protein export and sorting from the trans-Golgi network (TGN). However, accurately quantifying protein transport from one compartment to the next in the secretory pathway has been challenging. The Retention Using Selective Hooks (RUSH) system is a method that allows monitoring trafficking of a protein of interest in real time, similar to a pulse-chase experiment but without the need of radiolabeling. Accurate calculations, however, are necessary and currently lacking. Here, we combine the RUSH system with live cell imaging to quantify and calculate half lives. We exemplify our approach using a soluble secreted protein (LyzC). This system will benefit membrane trafficking researchers by adding numbers to protein export and comparing the export kinetics of different cargoes and variating conditions.

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Author information

Author notes

  1. Mai Ly Tran and Yeongho Kim contributed equally with all other contributors.

Authors and Affiliations

  1. Department of Cell Biology, Yale University School of Medicine, New Haven, CT, USA

    Mai Ly Tran, Yeongho Kim & Julia von Blume

Authors
  1. Mai Ly Tran
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  2. Yeongho Kim
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  3. Julia von Blume
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Corresponding author

Correspondence to Julia von Blume .

Editor information

Editors and Affiliations

  1. Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI, USA

    Yanzhuang Wang

  2. Department of Physiology and Cell Biology, University of Arkansas for Medical Sciences, Little Rock, AR, USA

    Vladimir V Lupashin

  3. Department of Biological Sciences, Vanderbilt University, Nashville, TN, USA

    Todd R. Graham

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Tran, M.L., Kim, Y., von Blume, J. (2023). Quantification of Protein Exit at the Trans-Golgi Network. In: Wang, Y., Lupashin, V.V., Graham, T.R. (eds) Golgi. Methods in Molecular Biology, vol 2557. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2639-9_35

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  • DOI: https://doi.org/10.1007/978-1-0716-2639-9_35

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2638-2

  • Online ISBN: 978-1-0716-2639-9

  • eBook Packages: Springer Protocols

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