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Vol. 9, Issue 8, 2201-2216, August 1998
-Tubulin
Complex
and§
The spindle pole body (SPB) in Saccharomyces
cerevisiae functions as the microtubule-organizing center.
Spc110p is an essential structural component of the SPB and spans
between the central and inner plaques of this multilamellar organelle.
The amino terminus of Spc110p faces the inner plaque, the substructure
from which spindle microtubules radiate. We have undertaken a synthetic
lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes
mutations in the amino terminus. The screen identified mutations in
SPC97 and SPC98, two genes encoding
components of the Tub4p complex in yeast. The spc98-63
allele is synthetic lethal only with spc110 alleles that
encode mutations in the N terminus of Spc110p. In contrast, the
spc97 alleles are synthetic lethal with
spc110 alleles that encode mutations in either the N
terminus or the C terminus. Using the two-hybrid assay, we show that
the interactions of Spc110p with Spc97p and Spc98p are not equivalent.
The N terminus of Spc110p displays a robust interaction with Spc98p in
two different two-hybrid assays, while the interaction between Spc97p
and Spc110p is not detectable in one strain and gives a weak signal in
the other. Extra copies of SPC98 enhance the interaction
between Spc97p and Spc110p, while extra copies of SPC97
interfere with the interaction between Spc98p and Spc110p. By testing
the interactions between mutant proteins, we show that the lethal
phenotype in spc98-63 spc110-221 cells is caused by
the failure of Spc98-63p to interact with Spc110-221p. In contrast,
the lethal phenotype in spc97-62 spc110-221 cells can
be attributed to a decreased interaction between Spc97-62p and Spc98p.
Together, these studies provide evidence that Spc110p directly links
the Tub4p complex to the SPB. Moreover, an interaction between Spc98p
and the amino-terminal region of Spc110p is a critical component of the
linkage, whereas the interaction between Spc97p and Spc110p is
dependent on Spc98p.
Department of Biochemistry and
Molecular and Cellular Biology Program, University of
Washington, Seattle, Washington 98195
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