Biomedical Engineering Reference
In-Depth Information
synthesis, Snc1 transits the ER and Golgi and travels in secretory
vesicles to the cell surface, where it aids in vesicle fusion with the
plasma membrane. It is then rapidly internalized and transported
back to the Golgi via an early endosomal recycling pathway, to
participate in further rounds of transport [
5
]. Because Snc1 does
not visit late endosomes, it can be used to identify genes required
specifi cally for early endosome sorting, in addition to those
involved in general endocytosis or exocytosis.
The localization of Snc1 in mutant strains is commonly moni-
tored by green fl uorescent protein (GFP) tagging and visualizing
by fl uorescence microscopy. While this approach has many advan-
tages, phenotypic assays that provide biochemical readouts are
typically more sensitive and quantitative and can lead to identifi ca-
tion of mutants with relatively weak traffi cking defects. Additionally,
biochemical assays can be conducted on a large scale and therefore
are well suited to screening yeast genome-wide mutant collections.
To identify mutants with altered endocytosis and recycling through
early endosomes, we created a Snc1-based chimeric reporter that
can be used to systematically screen yeast colony arrays using an
adapted invertase assay [
6
]. Biochemical approaches to study addi-
tional pathways, including traffi cking through late endosomes,
have been presented in the previous version of this chapter [
7
].
The GFP-Snc1-Suc2 (GSS) reporter contains Snc1 fused to GFP
on the cytosolic side as well as the invertase enzyme (encoded by the
SUC2
gene) on the extracellular side (
see
Fig.
1
). It is integrated into
the
SUC2
locus which disrupts endogenous invertase expression [
8
].
Invertase activity can be measured by a colorimetric assay using mem-
brane-impermeant reagents [
8
]. Thus, it provides a sensitive measure
of the amount of the reporter molecule on the cell surface.
Additionally, the modular nature of the construct means that Snc1
transmembrane or cytoplasmic sequences can easily be replaced by
sequences encoding different sorting signals to create new chimeric
reporters specifi c for other traffi cking pathways.
Here we have provided a detailed methodology for introduc-
ing the GSS reporter into desired strains and handling mutant
yeast arrays, followed by a description of two assays to quantita-
tively and qualitatively assess traffi cking of the GSS reporter. These
assays can be applied to study endocytosis, endosome recycling, as
well as secretory pathways.
2
Materials
2.1 Introducing
the GSS Reporter into
Yeast Strains
pHB4 GSS reporter (Addgene, 53462).
2.1.1 Plasmid
