mammalian Sec5 and Exo84 proteins [34–36] were perhaps later acquisitions. The similarity of the N-terminal modules is intriguing. With the exception of Exo70p, the N-terminal domains have not been soluble enough for biophysical and structural studies. They may be unstable, sticky, or perhaps natively unfolded in the absence of other exocyst subunits. It is tempting to speculate that the N-terminal domains might participate in protein-protein interactions at the core of the assembled exocyst, as seen in the electron micrographs of the bovine exocyst complex [29]. The C-terminal domains, therefore, might then play roles in interactions with small GTPases, the membrane, and other potential binding partners [3,4]. Because the exocyst structures comprise only domains of the proteins, we repeated the HMM predictions with the full-length sequences. We determined that all the exocyst subunits can be linked to each other (Table 2). The difference in P-values between Table 1 and Table 2 is due to the usage of the complete sequence versus only parts of it. These results corroborate the preliminary analyses by [11], implying that all members of the exocyst complex are sequence related, and indicating that all exocyst subunits have a related structure. Creation and purification of a soluble yeast Sec10p construct In order to understand more about the structure and function of the exocyst complex, it is imperative to have soluble purified subunits for structural studies and for reconstitution of the functional complex in vitro. Identification of a soluble structural domain for a previous insoluble exocyst subunit would validate our structure predictions, as well as provide soluble protein for further characterization. We chose to examine a predicted domain of the yeast Sec10p. Sec10 is one of the central subunits in the exocyst complex, and qualitative binding studies and yeast two-hybrid analyses indicate that it interacts with other exocyst subunits: Sec5, Sec6, Sec8, Sec15, Exo70 and Exo84 [3,12,15]. In addition, overexpression of either an N-terminal region (1–589) or a C-terminal region (590–872) results in dominant negative phenotypes in yeast [27]. Sec10p is one of the subunits that had previously been difficult to produce in a soluble recombinant form. We attempted a number of different strategies to produce soluble Sec10p protein. Initial efforts included the design of twenty different truncations using only secondary structure predictions. The N- and C-termini of these constructs were chosen to reside in non-structured regions so that predicted helices were not disrupted. Truncations were expressed with one of several affinity tags (e.g. His6, MBP and GST). The use of MBP as an N-terminal fusion tag appeared Table 2. Similarity between full-length exocyst subunits. Exocyst Helical Bundles promising, as milligrams of soluble Sec10p were produced. However, removal of the MBP tag by proteolytic cleavage resulted in immediate precipitation of Sec10p, suggesting that MBP was solubilizing misfolded and/or aggregated Sec10p protein; indeed, this problem has been previously observed with other proteins [37]. We also tried to co-express Sec10p and several truncations with either its partner exocyst subunit Sec15p [2] or with the chaperones GroEL/GroES [38]. These strategies did not improve the solubility of the Sec10p constructs (data not shown). Based on the HMM structure predictions described above, we identified N- and C-terminal ends of a putative Sec10p structural domain. The HMM analyses predicted a domain with similarity in the N-terminal region to the structure of Exo70p (in the range of 186–465) and in the C-terminal region to the structure of Sec6CT (range of 563–860). Based on this structural assignment, we designed a fragment that would encompass both domains. Consideration of the secondary structure prediction for this region (Figure 2A) led us to clone a construct from residues 145–827, containing a Pro at the N-terminus and a Gly at the C-terminus. This domain was predicted to contain four separate helical bundles, similar to those found in Exo70p and Sec6CT. This construct, Sec10(145–827), was cloned into the T7 expression vector pET15b. We chose to use an N-terminal His6 tag in order to maximize the likelihood of obtaining properly folded protein. Our previous experience using other constructs containing His6 tags is that this tag is not capable of solubilizing improperly folded recombinant proteins, but is useful for affinity purification. Upon overexpression in BL21(DE3) cells, this 145–827 Sec10p trunca- tion construct was found to be quite soluble, compared to other truncations, including 1–589, 590–871, 75–859, and 55–589 (Figure 2B). The Sec10(145–827) protein was expressed and purified using Ni-NTA chromatography, followed by size exclusion chromatog- raphy to remove several co-purifying contaminants (Figure 2B). Circular dichroism (CD) studies on the purified Sec10(145–827) protein indicate a predominantly helical protein (Figure 3A); calculations of apparent a-helicity suggest that Sec10(145–827) is approximately 60% helical [39], which is consistent with the ,60% helical content that we predicted. Moreover, purified Sec10(145–827) displays a symmetrical monodisperse gel filtration profile, which indicates a single well-folded protein species (Figure 3B). Function of Sec10(145–827) Because this construct of Sec10p appeared to be an indepen- dently folded structural domain, we tested to see if it was YJL085W YBR102C YIL068C YGL233W YER008C YDR166C YPR055W YLR166C Exo70p Exo84p Sec6p Sec15p Sec3p Sec5p Sec8p Sec10p Exo70 0 4.60E-02 1.20E-01 2.30E-02 9.10E-05 1.40E-01 4.10E-02 2.10E-06 Exo84 4.40E-02 0 1.90E-02 2.40E-02 1.10E-02 3.80E-06 2.90E-04 6.90E-03 Sec6 1.20E-01 2.00E-02 0 8.80E-02 1.10E-05 1.60E-02 5.40E-02 1.30E-02 Sec15 2.30E-02 2.50E-02 8.60E-02 0 1.60E-02 4.90E-07 6.40E-09 7.70E-04 Sec3 6.30E-05 1.00E-02 5.70E-06 1.40E-02 0 2.40E-03 3.60E-04 2.30E-05 Sec5 1.30E-01 2.10E-06 1.40E-02 2.10E-07 2.40E-03 0 4.40E-06 8.60E-05 Sec8 4.50E-02 3.80E-04 5.80E-02 1.00E-08 6.60E-04 1.10E-05 0 1.60E-02 Sec10 6.70E-07 4.40E-03 8.50E-03 3.60E-04 1.40E-05 5.20E-05 9.30E-03 0 HMM P-values of the comparisons are indicated for the full length proteins. SGD identifiers are indicated in the first column of the table. doi:10.1371/journal.pone.0004443.t002 PLoS ONE | www.plosone.org 4 February 2009 | Volume 4 | Issue 2 | e4443