Chapter title |
Large-scale analysis of membrane transport in yeast using invertase reporters.
|
---|---|
Chapter number | 27 |
Book title |
Membrane Trafficking
|
Published in |
Methods in molecular biology, January 2015
|
DOI | 10.1007/978-1-4939-2309-0_27 |
Pubmed ID | |
Book ISBNs |
978-1-4939-2308-3, 978-1-4939-2309-0
|
Authors |
Lauren Dalton, Michael Davey, Elizabeth Conibear, Dalton, Lauren, Davey, Michael, Conibear, Elizabeth |
Abstract |
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. |
X Demographics
As of 1 July 2024, you may notice a temporary increase in the numbers of X profiles with Unknown location. Click here to learn more.
Geographical breakdown
Country | Count | As % |
---|---|---|
France | 1 | 100% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 1 | 100% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 15 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Master | 3 | 20% |
Professor > Associate Professor | 2 | 13% |
Student > Ph. D. Student | 2 | 13% |
Student > Bachelor | 1 | 7% |
Professor | 1 | 7% |
Other | 4 | 27% |
Unknown | 2 | 13% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 7 | 47% |
Agricultural and Biological Sciences | 5 | 33% |
Unspecified | 1 | 7% |
Unknown | 2 | 13% |