Constitutive signaling by a mutant form of the amino acid sensor
Ssy1.
Richard F. Gaber, Kim E. Ottow, Helge A. Andersen,
Morten C. Kielland-Brandt
Department of Physiology, Carlsberg
Laboratory, Gl. Carlsberg Vej 10, Copenhagen Valby, DK-2500, Denmark
An emergent theme in nutrient sensing in S. cerevisiae is
the apparent evolution of transporters into sensors. We wish to better
understand how Ssy1 functions to initiate amino acid signaling and
developed a growth assay for Ssy1 signaling that is independent of amino
acid uptake. Briefly, a strain was constructed in which the genes for
the endogenous potassium transporters, TRK1 and TRK2 were
deleted and potassium uptake was made dependent on expression of the
Arabidopsis potassium channel, Kat1. By placing expression of the
KAT1 cDNA under the control of the amino acid-responsive promoter
from the AGP1 gene, we generated cells that grow on low
concentrations of potassium only in the presence of signaling amino
acids, e.g. Leu or Trp. This strain was used to screen for the effects
of PCR-induced mutations in SSY1. One mutant allele, SSY1-102, was identified that confers amino acid-independent expression
of the AGP1:KAT1 construct. Constitutive expression of an
AGP1:lacZ reporter confirmed the effect of the SSY1-102
allele. These results establish that amino acid transport is not an
essential feature of Ssy1-dependent signaling and thus strongly support
the notion that Ssy1 functions as a receptor that recognizes the
presence of extracellular amino acids. The nature and probable
functional role of the gain-of-function SSY1-102 allele will be
discussed. Northwestern University is acknowledged for granting a
sabbatical to R.F.G. at the Carlsberg Laboratory in 2000-2001.
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