Exocyst Helical Bundles Figure 2. Recombinant Sec10(145–827) is soluble. Several Sec10p truncation constructs designed using secondary structure predictions are not generally soluble. (A) Secondary structure prediction [41] and schematic of several representative N- and C-terminal truncations tested. The secondary structure prediction is schematically depicted as in Figure 1. Truncations 1–589 and 590–871 were derived from dominant negative constructs described previously [27]. (B) E. coli cells were transformed with Sec10p truncation variants cloned with an N-terminal His6-tag in the vector pET15b (Novagen). Expression was induced by addition of IPTG to 0.1 mM, and growth was continued at 15uC for 14–18 h. Cells were pelleted, lysed and the insoluble (P) material was separated from the soluble material (S) by centrifugation; these were run on a 10% SDS-PAGE gel and stained with Coomassie blue dye. Asterisks indicate the migration of each construct. For each construct except Sec10(145–827), very little of the His6-tagged protein was in the soluble fraction. Although the Sec10(75–859) construct initially appeared promising, it was sticky and aggregated after partial purification on Ni-NTA resin. The right hand lane contains Sec10(145–827) after purification by Ni-NTA resin and gel filtration chromatography. doi:10.1371/journal.pone.0004443.g002 functional. We performed in vitro binding experiments with other purified exocyst subunits that bind Sec10p: Sec6p, Exo70p and Exo84p. Sec6p and Exo70p interactions with full-length Sec10p were previously shown by qualitative pull-down binding assays using partially purified proteins [12,15], while binding of Exo84p had only been observed in yeast two-hybrid assays [28]. For Exo70p (a.a. 63–623) and Exo84CT (a.a. 523–753), we used soluble truncations that had been determined by limited proteolysis and whose structures had previously been determined [12]. Sec6p, Exo70p and Exo84CT were N-terminally tagged with Maltose Binding Protein (MBP) for use in qualitative pull-down experiments. When the purified Sec10(145–827) protein was incubated for 1 h with MBP alone, or with the MBP-tagged proteins, it was found to bind specifically to MBP-Sec6p, -Exo70p and -Exo84CT, above background binding to MBP alone (Figure 4). Therefore, we conclude that Sec10(145–827) is properly folded and contains the binding domain(s) for Sec6p, Exo70p and the C-terminal domain of Exo84p. Remarkably, under our conditions, only about 10–20% of each recombinant exocyst subunit appears to interact with each other in in vitro binding experiments. Similar results have also been observed for the full-length proteins [12,15]. One possibility for PLoS ONE | www.plosone.org 5 February 2009 | Volume 4 | Issue 2 | e4443