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Vol. 12, Issue 6, 1645-1669, June 2001
*Department of Molecular and Cell Biology, Division of Biochemistry
and Molecular Biology, and §Electron Microscopy
Laboratory, University of California, Berkeley, California 94720
In Saccharomyces cerevisiae, entry into
mitosis requires activation of the cyclin-dependent kinase Cdc28
in its cyclin B (Clb)-associated form. Clb-bound Cdc28 is susceptible
to inhibitory tyrosine phosphorylation by Swe1 protein kinase. Swe1 is
itself negatively regulated by Hsl1, a Nim1-related protein kinase, and
by Hsl7, a presumptive protein-arginine methyltransferase. In vivo all
three proteins localize to the bud neck in a septin-dependent manner,
consistent with our previous proposal that formation of Hsl1-Hsl7-Swe1
complexes constitutes a checkpoint that monitors septin assembly. We
show here that Hsl7 is phosphorylated by Hsl1 in immune-complex kinase assays and can physically associate in vitro with either Hsl1 or Swe1
in the absence of any other yeast proteins. With the use of both the
two-hybrid method and in vitro binding assays, we found that Hsl7
contains distinct binding sites for Hsl1 and Swe1. A differential
interaction trap approach was used to isolate four single-site
substitution mutations in Hsl7, which cluster within a discrete region
of its N-terminal domain, that are specifically defective in binding
Hsl1. When expressed in hsl7
cells, each of these
Hsl7 point mutants is unable to localize at the bud neck and cannot
mediate down-regulation of Swe1, but retains other functions of Hsl7,
including oligomerization and association with Swe1. GFP-fusions of
these Hsl1-binding defective Hsl7 proteins localize as a bright
perinuclear dot, but never localize to the bud neck; likewise, in
hsl1 cells, a GFP-fusion to wild-type Hsl7 or native
Hsl7 localizes to this dot. Cell synchronization studies showed that,
normally, Hsl7 localizes to the dot, but only in cells in the G1 phase
of the cell cycle. Immunofluorescence analysis and immunoelectron
microscopy established that the dot corresponds to the outer plaque of
the spindle pole body (SPB). These data demonstrate that association
between Hsl1 and Hsl7 at the bud neck is required to alleviate
Swe1-imposed G2-M delay. Hsl7 localization at the SPB during G1 may
play some additional role in fine-tuning the coordination between
nuclear and cortical events before mitosis.
These authors contributed equally to this work.
Present address: Department of Microbiology II,
University of Madrid Complutense, 28040 Madrid, Spain.
Corresponding author. E-mail address:
jeremy{at}socrates.berkeley.edu.
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