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Vol. 13, Issue 4, 1144-1157, April 2002
-Tubulin
Complex
§ and
*Departments of Biochemistry and Nucleation of microtubules is central to assembly of the mitotic
spindle, which is required for each cell division. Physiology and
Biophysics, University of Washington, Seattle, Washington 98195; and
Department of Bioengineering, Pennsylvania State
University, University Park, Pennsylvania 16802
-Tubulin is a
universal component essential for microtubule nucleation from
centrosomes. To elucidate the mechanism of microtubule nucleation in
budding yeast we reconstituted and characterized the yeast -tubulin
complex (Tub4p complex) produced in insect cells. The recombinant
complex has the same sedimentation coefficient (11.6 S) as the native
complex in yeast cell extracts and contains one molecule of Spc97p, one
molecule of Spc98p, and two molecules of Tub4p. The reconstituted Tub4p
complex binds preformed microtubules and has a low nucleating activity,
allowing us to begin a detailed analysis of conditions that enhance
this nucleating activity. We tested whether binding of the recombinant
Tub4p complex to the spindle pole body docking protein Spc110p affects
its nucleating activity. The solubility of recombinant Spc110p in
insect cells is improved by coexpression with yeast calmodulin (Cmd1p).
The Spc110p/Cmd1p complex has a small sedimentation coefficient (4.2 S)
and a large Stokes radius (14.3 nm), indicative of an elongated structure. The Tub4p complex binds Spc110p/Cmd1p via Spc98p and the
Kd for binding is 150 nM. The low nucleation
activity of the Tub4p complex is not enhanced when it is bound to
Spc110p/Cmd1p, suggesting that it requires additional components or
modifications to achieve robust activity. Finally, we report the
identification of a large 22 S Tub4p complex in yeast extract that
contains multimers of Spc97p similar to -tubulin ring complexes
found in higher eukaryotic cells.
Corresponding author. E-mail address:
tdavis{at}u.washington.edu.
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