Exocyst Helical Bundles Figure 3. Sec10(145–827) is folded and a-helical. (A) The far-UV CD wavelength spectrum of Sec10(145–827) was measured between 200 and 270 nm at 4uC. The characteristic minimum at 222 nm is indicative of ,60% helicity. (B) Gel filtration profile of Sec10(145–827). Purified Sec10(145– 827) was applied to a Superdex 200 gel filtration column and the absorbance was monitored at 280 nm. The retention volume of molecular weight standards (in kD) are indicated at the top. Sec10(145–827) elutes in a single, monodisperse peak, although the apparent molecular weight of Sec10(145–827), based on the MW standards, indicates that it elutes slightly smaller than expected, suggesting deviation from a spherical shape, or a small amount of reversible non-specific interaction with the column. doi:10.1371/journal.pone.0004443.g003 Figure 4. Sec10(145–827) is functional for protein-protein interactions in vitro. Sec10(145–827) binds to MBP-Sec6p, MBP-Exo70p (residues 63–623) and MBP-Exo84p (residues 523–753), but not to MBP alone. The MBP, MBP-tagged Sec6p, Exo70p and Exo84p proteins were immobilized on amylose resin and incubated with Sec10(145–827). Equivalent volumes of the bound fractions [10% of the input of Sec10(145–827) is shown in the first lane as a control for the amount of Sec10(145–827) bound] were analyzed on denaturing SDS-PAGE gels. His6-tagged Sec10(145–827) and MBP- tagged partners were detected by Western blot analyses using a-His5 and a-MBP antibodies, respectively. doi:10.1371/journal.pone.0004443.g004 PLoS ONE | www.plosone.org 6 February 2009 | Volume 4 | Issue 2 | e4443