Regulation of the Fab1 PI3P-5 kinase, a lipid kinase that functions
in vacuolar protein sorting and vacuolar size control.
Simon
A. Rudge (1), Deborah Anderson (1), Trey Sato (1), Jonathan D. Gary
(1), Lois S. Weisman (2), Scott D. Emr (1)
(1) HowardHughes Medical
Institute, UCSD, 9500 Gilman Drive, La Jolla, CA 92093-0668, USA; (2)
Department of Biochemistry, University of Iowa, Iowa City, IA
52242.USA
FAB1 encodes a Type III PIP kinase that synthesizes
PI3,5P2. The major phenotypes resulting from Fab1 kinase
inactivation and loss of cellular PI3,5P2 include, a
temperature-dependent growth defect, dramatic increases in vacuolar size
and the failure to sort specific cargo molecules into late endosomes
(multi-vesicular bodies, MVBs). Interestingly, deletion of VAC7,
which encodes a 128 Kda vacuolar transmembrane protein, also results in
the loss of PI3,5P2 as well as the other fab1 defects.
Since Vac7 does not contain a PIP kinase domain, this result suggests
that Vac7 functions as a candidate regulator of the Fab1 kinase. To
test this model, we mutagenized FAB1 in an attempt to bypass the
requirement for Vac7, and undertook a genetic screen to identify
vac7 bypass suppressors. We isolated one mutant allele of
FAB1, fab1-5, and our genetic screen identified a mutant
allele of FIG4, fig4-1, whose gene product contains a Sac1
polyphosphoinositide phosphatase domain. Expression of either fab1-5 or deletion of FIG4 in a vac7 deletion mutant,
suppressed the temperature-sensitive growth and the vacuolar defects
associated with deleting VAC7. In addition, PI3,5P2
synthesis was also dramatically restored. These results suggest that
synthesis of PI3,5P2 by Fab1 kinase is necessary for MVB
sorting and is regulated by Vac7, while turnover of PI3,5P2
is mediated in part by Fig4. Present efforts are focussed on identifying
PI3,5P2-specific effector proteins.
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