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ISABEL ÁLVAREZ-TABARÉS Y COLS. ANAL. REAL ACAD. NAC. FARM.

with a synthetic peptide encompassing the RVSF sequence
established the RVXF motif as a conserved binding sequence that
associates with a hydrophobic pocket on the surface of the PP1
catalytic subunit (19). Recently a systematic analysis of structural
elements that mediate the binding specificity of PP1 interacting
proteins proposed a refined consensus motif for high affinity PP1
ligands (20). Application of the results of this study to protein
sequence databases searches enabled the authors to predict PP1-
interacting partners and mutational analysis have demonstrated that
differences in peptide-protein interactions dictate the affinity of PP1
for cellular regulators and control the dynamic physiological
regulation of PP1 functions in the cell.

PP1 IN S. pombe

While mammalian cells contain four isoforms of the PP1 catalytic
subunit (a, ß, ? and d), fission yeast contains two (6, 21). The
unicellular lifestyle, sophisticated genetics and systematic approaches
to study phosphorylation (22) makes yeasts particularly attractive
models to study PP1 function (23).

The S. pombe genome encodes two highly related PP1 catalytic
subunits Dis2 and Sds21. PP1 localisation was determined by fusing
sequences encoding EGFP to the N-termini of Dis2 and Sds21. In
order to maximise the likelihood that the tagged proteins were
expressed at their normal physiological levels, the «marker switch
approach» (24) to integrate the manipulated versions of dis2 and
sds21 genes at their native loci was used. Either gene can be
individually deleted, however, simultaneous deletion of both is lethal
(21, 25). Importantly, both N-terminally EGFP-tagged proteins were
shown to be functional as ?sds21 cells expressing a Dis2.NEGFP at
the dis2+ locus and ?dis2 cells expressing Sds21.NEGFP at the sds21+
locus were viable and did not show any apparent defect in
morphology, generation time and the actin and microtubule
cytoskeletons.

PP1 has been localised to the nucleus in all organisms studied to
date, such as S. cerevisiae, S. pombe, Aspergillus nidulans, and
mammalian cells. Consistent with these previous studies, Dis2.

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