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Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen

Nature Protocols volume 1, pages 2439–2447 (2006)Cite this article

Abstract

Secreted and cell surface proteins play essential roles in numerous essential biological processes in eukaryotic organisms, but are often more difficult to isolate and identify than proteins that are localized in intracellular compartments. However, several high-throughput 'gene-trap' techniques have been developed to characterize these 'secretomes', including the yeast secretion trap (YST) screen. This method involves fusing cDNA libraries from the tissue or cell type of interest to a yeast (Saccharomyces cerevisiae) invertase reporter gene, transforming the resulting fusion library into an invertase-deficient yeast strain and plating the transformants on a medium containing sucrose as the sole carbon source. A yeast cell with a transgene encoding a secreted or cell surface protein can synthesize a secreted invertase fusion protein that can rescue the mutant, and the plasmid DNA can then be sequenced to identify the gene that encodes it. We describe a recently improved version of this screen, which allows the identification of genes encoding secreted proteins in 1–2 months.

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Figure 1: Schematic diagram of the basic steps in the yeast secretion trap (YST) system to identify eukaryotic secreted and cell surface proteins.
Figure 2: Schematic diagram of the pYST vector system used for cDNA library construction.

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Acknowledgements

Research in this area was supported by grants from the US National Science Foundation Plant Genome Program (DBI-0606595) and the New York State Office of Science, Technology and Academic Research (NYSTAR). S.-J.L. and B.-D.K. of the Center for Plant Molecular Genetics and Breeding Research were also supported by a grant from KOSEF/MOST. The authors would like to thank O.K. Park (Graduate School of Biotechnology, Korea University, Seoul, Korea) for the generous gift of the yeast strain DBYα2445 and T. Isaacson for careful reading of this manuscript.

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Authors and Affiliations

  1. Department of Plant Biology, 228 Plant Sciences Building, Cornell University, Ithaca, 14853, New York, USA

    Sang-Jik Lee & Jocelyn K C Rose

  2. Center for Plant Molecular Genetics and Breeding Research, Seoul National University, Seoul, 151-921, Korea

    Sang-Jik Lee & Byung-Dong Kim

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Correspondence to Jocelyn K C Rose.

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Lee, SJ., Kim, BD. & Rose, J. Identification of eukaryotic secreted and cell surface proteins using the yeast secretion trap screen. Nat Protoc 1, 2439–2447 (2006). https://doi.org/10.1038/nprot.2006.373

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