Biomedical Engineering Reference
In-Depth Information
Chapter 27
Large-Scale Analysis of Membrane Transport in Yeast
Using Invertase Reporters
Lauren Dalton , Michael Davey , and Elizabeth Conibear
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 meth-
ods. The fi rst 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 intro-
duced, 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 traffi cking
pathways.
Key words
Yeast, Deletion mutant collection, Phenotypic screening, Vesicle transport, Golgi,
Endosome, Endocytosis, Snc1, GSS, Invertase
1
Introduction
Newly synthesized proteins, translocated into the ER, are transported
to various membrane-bound organelles, where they perform their
specifi c functions. These transport processes are tightly regulated
and require the concerted action of vesicle coat proteins and other
regulatory factors [
1
-
3
]. When a particular traffi cking step is dis-
rupted, proteins requiring that pathway are mislocalized. Thus, we
can use protein mislocalization as a method to identify and study
regulators of specifi c traffi cking pathways.
Snc1, a membrane-associated protein related to mammalian
synaptobrevin, is a well-established model protein used to study
endocytic recycling in the yeast
Saccharomyces cerevisiae
[
4
]. After
