Page 97 - 73_04

VOL. 73 (4), 901-925, 2007 ROLES OF PROTEIN PHOSPHATASE TYPE 1...

NEGFP and Sds21.NEGFP were greatly enriched in the nucleus,
being Sds21.NEGFP concentrated in the nucleolus (Figure 2A, B).
Intriguingly, in addition to the anticipated nuclear localisation, new
locations for Dis2.NEGFP, but not for Sds21.NEGFP, were also
observed. Dis2.NEGFP appeared as a bright dot at the nuclear
periphery, at the cell tips, endocytotic vesicles and in a ring at the
cell equator in early anaphase (Figure 2A, inset). Deletion of dis2+
from sds21.NEGFP cells led to an increase in Sds21.EGFPN protein
levels and the incorporation of Sds21.NEGFP into all of the locations
normally occupied by Dis2, with the exception of chromatin and the
staining around the cell equator during division (Figure 2C).

The bright nuclear dot of Dis2.NEGFP colocalised with a
centromeric marker, Cnp1.Cherry, and chromatin immunopre-
cipitation showed that Dis2.Npk associated with centromeric
sequences of the central core non-repetitice domains of the
centromeres (Figure 2D).

The cytoplasmic Dis2.NEGFP foci could be divided into two
distint subpopulations. One appearing at the cell cortex and moving
from it into the cytoplasm for a short distance and another one
associated with the cell tips (Figure 3A). The internalisation was
dependent on a functional F-actin cytoskeleton and on the
endocytotic factor Sla2. The recruitment to cell tips was dependent
on the S. pombe Bud14 homologue, Wsh3/Tea4 (26, 27) (Figure 3B),
and the Kelch domain protein Tea1 (Figure 3C). Wsh3/Tea4 co-
localised with Dis2 and physically associated with it (Figure 3D, E).
This association relied upon the conserved PP1 binding consensus
site in Wsh3/Tea4 (RV223XF225) (Figure 3E). Mutation of this sequence
blocked the recruitment of Dis2 to cell tips, but neither Tea1 nor
Wsh3/Tea4 itself. Moreover, mutation of the PP1 binding site of
Wsh3/Tea4 compromised the control over the establishment and
choice of polarised tip growth that accompanies cell cycle
progression of unperturbed cultures and led to a major deficiency in
re-establishing polarised growth from existing tips following osmotic
stress. This function may be due, in part, to the impact of Wsh3/
Tea4 upon actin polymerisation as the ability of excess Wsh3/Tea4
to induce excessive F-actin cables was abolished by mutation of the
PP1 binding sites (28).

907