Large-Scale Analysis of Membrane Transport in Yeast Using Invertase Reporters - Membrane Trafficking - page 400

Horseradish Peroxidase (HRP, Sigma-Aldrich, P6782) 1 mg/
mL in dH 2 O. Filter sterilize. Store at 4 °C.

●

N -Ethylmaleimide (NEM, Sigma-Aldrich, E1271): 20 mM in
dH 2 O. Prepare NEM immediately prior to the assay.

●

O -Dianisidine (Sigma-Aldrich, D3252): 10 mg/mL in
dH 2 O. Prepare immediately prior to the assay.

●

3 Methods

3.1 Introducing
the GSS Reporter
and Handling Yeast
Arrays

A schematic diagram of the GSS plasmid is shown in Fig. 2 . The
expression cassette consists of an ADH promoter driving the GFP-
Snc1-Suc2 chimera, preceded by the ClonNat R selectable marker,
and fl anked by sequences corresponding to 5 regions of

SUC2 . In order to perform the invertase assay, the GSS reporter is
fi rst introduced into strain(s) of interest that do not express endog-
enous SUC2, which would otherwise have background invertase
activity. Because GSS is a centromere-based plasmid with both
URA3 and ClonNat R selectable markers, it can be maintained at
single copy in suc2

′ and 3 ′

strains in media that either lacks uracil or con-
tains Nat. Alternatively, the cassette containing the GSS reporter
and ClonNat R selectable marker can be excised and integrated at
the SUC2 locus by homologous recombination, thus disrupting
the expression of wild-type SUC2 .
An important feature of the plasmid is the presence of unique
restriction sites that allow different parts of the chimera to be
exchanged by homologous recombination. This makes it easy to
create chimeras containing different transport signals or using
different promoter sequences to drive expression. For example, we
have created reporters in which Snc1's endocytosis signal has been
mutated or where the entire Snc1 cytosolic domain has been
replaced by that of the t-SNARE protein Sso1, which is internal-
ized by different adaptor proteins [ 6 ].

∆

Fig. 2 GSS reporter plasmid map: The GSS reporter plasmid expresses a GFP-Snc1-Suc2 chimera driven by
the ADH promoter. Surrounding the chimera are regions of fl anking homology to SUC2 . The chimera and
reporter can be excised through a NotI/SnaBI double digest and integrated at the SUC2 locus. Additionally,
unique restriction sites allow the exchange of the promoter and chimera components by homologous recom-
bination. Illustration is not to scale