Biology Reference
In-Depth Information
2 Use of Yeast to Study Mammalian Proteins
As unicellular eukaryotes, the budding yeast
Saccharomyces cerevisiae
and the
fission yeast
Schizosaccharomyces pombe
serve as important model organisms for
the study of biological processes that are conserved in human cells. Both yeasts have
been used to clone human genes by functional complementation of a defective gene
in the host yeast strain. For example, the human Cdc2 cyclin-dependent kinase gene
was first cloned by its ability to suppress the temperature-sensitive growth of a
S.
pombe cdc2
mutant strain (Lee and Nurse
1987
). Yeast strains expressing human
proteins are used to study the function of the human protein and to identify mutations
that alter function, as in a study of the p53 protein expressed in
S. pombe
(Bischoff
et al.
1992
). Furthermore, yeast growth-based High-Throughput Screens (HTSs)
have been successfully deployed to identify inhibitors of various eukaryotic pro-
teins, including sirtuin family NAD-dependent deacetylases (Grozinger et al.
2001
),
mammalian K
+
channels (Zaks-Makhina et al.
2004
), and mammalian p38
a
MAP
kinase (Friedmann et al.
2006
).
Studies using
S. cerevisiae
first demonstrated the feasibility of cloning and
studying mammalian PDE genes in a yeast system.
S. cerevisiae
expresses two
PDE proteins, the Pde1 low-affinity enzyme, and the Pde2 high-affinity enzyme
(Nikawa et al.
1987b
; Sass et al.
1986
). Cells lacking Pde1 and Pde2 have high
cAMP levels that confer heat-shock sensitivity to stationary phase cells. Library
screens for mammalian genes that restore heat-shock resistance led to the identifi-
cation of clones of rat PDE4B and human PDE7A (Colicelli et al.
1989
,
1991
;
Michaeli et al.
1993
). In addition, yeast strains that express mammalian PDE4B
display a heat-shock survival phenotype that is sensitive to treatment with the PDE4
specific inhibitor, rolipram (Engels et al.
1995
; McHale et al.
1991
; Pillai et al.
1993
,
1994
; Torphy et al.
1992
). The ability to monitor PDE4 activity via heat shock
resistance and the sensitivity of yeast-expressed rat PDE4B to rolipram allowed for
the isolation of rolipram-resistant forms of PDE4B (Pillai et al.
1993
). The amino
acids altered in these proteins were later shown to be part of the nucleotide-binding
site in the active site. While this system facilitated the cloning and study of PDEs, it
is not amenable to high-throughput screening as abnormally high concentrations of
rolipram are required for this assay. This may be due to the need to work with
stationary phase cells that are relatively impenetrable to test compounds.
3
cAMP Signaling and
fbp1
Transcriptional Regulation
in the Fission Yeast
S. pombe
Both
S. pombe
and
S. cerevisiae
produce cAMP signals in response to glucose
detection (D'Souza and Heitman
2001
; Hoffman
2005a
,
b
; Lengeler et al.
2000
;
Thevelein et al.
2000
; Thevelein and de Winde
1999
). In these yeasts, the increase
in cAMP levels is due to adenylyl cyclase activation, while feedback regulation to
