[2,12,15]. The specific function(s) of Sec10p is unknown; however, overexpression of N- and C-terminal truncated constructs show dominant negative secretory and morphogenic defects in vivo [27]. Further characterization of Sec10p by biophysical and structural methods has been hindered by the lack of soluble recombinant Sec10p protein. We expressed and purified the predicted Sec10p structural domain, and show that it is folded and helical in solution. This domain is functional—it retains the ability to interact with both Sec6p and Exo70p. In addition, we show that it interacts directly with the C-terminal domain of Exo84p, an interaction previously shown only by yeast two-hybrid studies [28]. Thus, our bioinformatic analyses have revealed structural similarity between all exocyst components and have additionally defined a soluble domain of the exocyst complex subunit Sec10p for further biochemical and structural characterization. Results and Discussion All of the exocyst subunits have similar helical bundle structures The exocyst complex is composed of eight large proteins (between 71 and 155 kD) that are predicted to be predominantly helical by secondary structure predictions (Figure 1B). They form a complex at sites of exocytosis, have been proposed to tether secretory vesicles to the plasma membrane, and may serve as a quality control mechanism to ensure proper membrane fusion [3,4]. Little is known about the structure of the intact complex, except a series of images taken by quick freeze-deep etch EM [29]. With less than 10% sequence identity between them, the exocyst subunits were originally thought to be unrelated. However, several short stretches in each of the subunits are predicted to form coiled coil (or amphipathic helical) structures [1,7,30], and regions of similarity with subunits from other tethering complexes have been detected [7,11,30]. When the high resolution crystal structures of several exocyst subunits (Sec15p, Exo70p, Exo84CT, and Sec6CT) were determined, it became clear that they are structurally and topologically similar (Figure 1A; [3]). Searches of the Protein Data Bank (PDB; [31] using Dali (http://www.ebi. ac.uk/dali) indicated that these helical bundle structures are considerably more similar to each other than to other proteins. The structures of the cargo binding domain of Myo2p, the unconventional type V myosin that transports yeast secretory vesicles [32], as well as the Conserved Oligomeric Golgi tethering Table 1. Similarity to exocyst subunits of known structures. Length(aa) scExo70p YJL085W YBR102C YIL068C YGL233 YER008C YDR166C YPR055W YLR166C Exo70p Exo84p Sec6p Sec15p Sec3p Sec5p Sec8p Sec10p 622 752 804 909 970 1335 870 1064 Exocyst Helical Bundles complex subunit, COG2 (residues 61–262; [9]), also show structural similarity to the exocyst helical bundles. It is unclear whether the similarity of COG2 represents divergence or functional convergence of the COG complex subunits from the exocyst subunits [7,9,11,30]. The structural relatedness between the different exocyst subunits, combined with similar patterns of predicted helical secondary structures (Figure 1; also [12,15,16]) led us to predict that all the exocyst structures would be similar, and to create a new working model for the exocyst complex structure [3]. This structural similarity was recently supported using multiple iterations of PSI-BLAST [33], for four of the exocyst subunits [11]. Here, we use hidden Markov models to examine the relatedness of the exocyst subunits at the sequence level. These analyses use comparisons of different HMM profiles generated from the individual exocyst families combined with secondary structure predictions and the known structures; this method has previously been shown to be more sensitive than other current analyses [26]. Indeed, statistically significant P-values were detected between all the exocyst components of known structure (Table 1). Two families of exocyst structures can be distinguished in the helical bundle structures of the exocyst components [not including the Ral binding domains of mammalian Sec5 [34,35] and Exo84 [36]]: the Exo70/84 and Sec6/15 families. The Exo70 (2B1E_A & 2PFT_A) HMM detects the Exo84 (2D2S_A) HMM with a P- value of 1024 and reciprocally, while the Sec6 (2FJI_1) and Sec15 (2A2F_X) HMMs detect each other with 1023 and 1027 P-values. All exocyst sequences of unknown structures can be linked to at least one exocyst sequence of known structure (Table 1). The exocyst subunits show a striking pattern with many of their N-terminal regions detecting the Exo70/84 families, and many of their C-terminal regions detecting the Sec6/15 families (Table 1). The exceptions are Exo84 and Sec3, which differ in their N- terminal regions; both contain coiled coil and b-sheet domains. The similarities suggest that the Sec3/5/8/10 proteins are formed by tinkering with modules based on the exocyst families of known structures: N-terminal modules are derived from the Exo70/84 families and C-terminal modules from the Sec6/15 families. These analyses suggest an ancient gene duplication event, followed by fusion of these genes and divergence, then followed by multiple gene duplication events and divergence to create the different subunits. Additional modules, such as the coiled coil and b-sheet regions of Sec3p and Exo84p and the Ral-binding domains of the 2.1E-04 (94–288) 1.7E-01 (12–342) 6.2E-03 (241–383) 2b1e_A 0 (60–623) 3.8E-03 (549–747) 3.0E-01 (98–666) 2.6E-02 (159–684) 9.4E-01 (221–660) 1.3E-01 (157–686) 1.3E-02 (187–695) musExo70 2pft_A 0 (70–622) 8.5E-04 (549–707) 5.1E-01 (361–802) 2.7E-01 (432–746) 4.4E-01 (153–551) 7.7E-02 (404–677) 3.1E-03 (186–465) Sec6CT 2fji_1 Exo84CT 2d2s_A 0 (523–753) 2.5E-01 (174–385) 0 (407–805) 4.6E-02 (81–252) 4.2E-03 (491–861) 2.2E-06 (228–378) 2.1E-04 (687–937) 1.4E-05 (161–228) 7.7E-03 (590–997) 1.7E-03 (178–393) 2.0E-04 (563–860) 1.0E-07 (720–1333) 2.6E-08 (733–1329) 4.7E-03 (1046–1305) dmSec15CT 2a2f_X 4.8E-07 (413–728) 0 (475–836) 4.8E-08 (625–920) 3.6E-07 (864–967) 1.4E-06 (578–826) HMM P-values of the comparisons are shown in bold, with the range of the aligned residues in parentheses below. Yeast protein lengths are indicated by the number of amino acids (aa). SGD identifiers are indicated in the first column of the table, and PDB identifiers are indicated in the second row of the table. Blank cells have P- values.1. doi:10.1371/journal.pone.0004443.t001 PLoS ONE | www.plosone.org 3 February 2009 | Volume 4 | Issue 2 | e4443