Large-Scale Analysis of Membrane Transport in Yeast Using Invertase Reporters

  • Lauren Dalton
  • Michael Davey
  • Elizabeth ConibearEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1270)


Transport of membrane proteins between cellular organelles requires the concerted action of many regulatory factors, which aid in cargo recognition and vesicle formation, targeting, and fusion. The yeast Saccharomyces cerevisiae is a useful model system for studying such regulators, due to the availability of genome-wide mutant collections and reporter proteins that provide sensitive biochemical readouts of individual transport pathways. Here, we describe an enzymatic invertase assay for evaluating endocytic recycling using a chimeric GFP-Snc1-Suc2 reporter. Cell surface levels of this reporter can be measured by a colorimetric assay that monitors sucrose hydrolysis at the plasma membrane, using two different methods. The first is a semiquantitative agar overlay assay followed by image densitometry that is suitable for high-throughput screening of arrayed yeast colonies. In the second, more quantitative assay, an enzymatic solution is added to yeast cultures in a multi-well plate and the absorbance is assessed by a plate reader. Furthermore, the modular nature of the chimeric reporter allows alternate transport signals to be introduced, thereby expanding the range of transport pathways that can be evaluated by this method. Together these techniques can be used to explore the function of genes involved in a variety of cellular trafficking pathways.

Key words

Yeast Deletion mutant collection Phenotypic screening Vesicle transport Golgi Endosome Endocytosis Snc1 GSS Invertase 


  1. 1.
    Keller P, Simons K (1997) Post-Golgi biosynthetic trafficking. J Cell Sci 110:3001–3009PubMedGoogle Scholar
  2. 2.
    Lemmon SK, Traub LM (2000) Sorting in the endosomal system in yeast and animal cells. Curr Opin Cell Biol 12:457–466CrossRefPubMedGoogle Scholar
  3. 3.
    van der Goot FG, Gruenberg J (2006) Intra-endosomal membrane traffic. Trends Cell Biol 16:514–521CrossRefPubMedGoogle Scholar
  4. 4.
    Gerst JE, Rodgers L, Riggs M, Wigler M (1992) SNC1, a yeast homolog of the synaptic vesicle-associated membrane protein/synaptobrevin gene family: genetic interactions with the RAS and CAP genes. Proc Natl Acad Sci U S A 89(10):4338–4342CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Lewis MJ, Nichols BJ, Prescianotto-Baschong C, Riezman H, Pelham HR (2000) Specific retrieval of the exocytic SNARE Snc1p from early yeast endosomes. Mol Biol Cell 11(1):23–38CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Burston HE, Maldonado-Báez L, Davey M, Montpetit B, Schluter C, Wendland B, Conibear E (2009) Regulators of yeast endocytosis identified by systematic quantitative analysis. J Cell Biol 185(6):1097–1110CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Burston HE, Davey M, Conibear E (2008) Genome-wide analysis of membrane transport using yeast knockout arrays. In: Vancura A (ed) Methods in molecular biology, vol 457. Humana Press, Totowa, NJ, pp 29–39Google Scholar
  8. 8.
    Darsow T, Odorizzi G, Emr SD (2000) Invertase fusion proteins for analysis of protein trafficking in yeast. Methods Enzymol 327:95–106CrossRefPubMedGoogle Scholar
  9. 9.
    Tong AHY, Boone C (2006) Synthetic genetic array analysis in Saccharomyces cerevisiae. Methods Mol Biol 313:171–192PubMedGoogle Scholar
  10. 10.
    Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Lauren Dalton
    • 1
  • Michael Davey
    • 1
  • Elizabeth Conibear
    • 1
    Email author
  1. 1.Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Child and Family Research InstituteUniversity of British ColumbiaVancouverCanada

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