The exocyst is a large, eight protein complex localized to sites of polarized secretion that is required for exocytosis and cytokinesis in eukaryotes (
; and references therein). Its specific function(s) is unclear, but it interacts with small Ras superfamily GTPases on secretory vesicles and the plasma membrane, where it is hypothesized to tether vesicles to the plasma membrane prior to membrane fusion 
. The complex also interacts with the regulatory protein, Sec1p 
, and the plasma membrane SNARE (soluble N-ethylmaleimide sensitive protein receptor) protein Sec9p 
. These interactions indicate a role for the exocyst and its subunits in the quality control of exocytic trafficking, as well as in facilitating SNARE complex assembly and vesicle fusion at the plasma membrane. Elucidation of the exocyst's function, and that of other related tethering complexes 
, requires biochemical and structural analyses of the individual subunits, as well as various protein-protein interactions within the complex.
Each of the exocyst subunits is predicted to be α-helical. They possess less than 10% sequence identity with each other, although limited sequence similarity has been detected using PSI-BLAST analyses 
. They also show little similarity to other proteins or domains, except for short regions of predicted coiled coils 
. Several recent crystal structures of domains from individual subunits have been determined: nearly full-length yeast and human Exo70 
, and the C-terminal domains of yeast Exo84p 
, yeast Sec6p 
. They show similar structures containing multiple helical bundles, yielding an overall similar shape (). Specific details of the bundles differ, especially the surface residues, but the helical bundle topologies are identical, suggesting divergent evolution from an ancient exocyst ancestor protein for these four exocyst components 
The exocyst subunits have similar helical bundle structures.
Progress for the other subunits has been hindered by lack of soluble protein. Insolubility can occur for many different reasons: recombinant proteins may not fold correctly when overexpressed in Escherichia coli
cells, they may not have the correct post-translational modifications, or they may be insoluble in the absence of co-factors or binding partners. Much effort has been spent to develop methods to address these issues (
; and references therein), including the use of different strains, selective growth conditions, fusion tags, co-expression with binding partners, and expression of independently folded structural domains. Often, the latter strategy is approached by using secondary structure predictions to deduce a domain, making numerous constructs with slight variations at the N- and/or C-termini, or by using limited proteolytic digestions to cleave unstructured or floppy regions, thereby defining a core domain. Limited proteolysis proved critical for most of the current exocyst structures; however, this strategy relies on availability of at least slightly soluble protein. An alternative approach is to computationally predict domain structures based on the similarity to proteins with known structural domains 
. However, this approach is challenging if the protein has little or no similarity with proteins of known structures. In that case, more sensitive computational methods, such as hidden Markov model (HMM) predictions 
, may be successful.
Based on the structural conservation observed in the exocyst subunits, we hypothesized that the other subunits would have similar helical bundle structures 
. Therefore, we examined the conservation of structural similarity between the subunits by profile HMM analyses using the HHSearch program 
. Profile HMMs are similar to simpler sequence profiles, but in addition to the amino acid frequencies in the columns of a multiple sequence alignment, they contain information about the frequency of inserts and deletions at each column, plus transition probabilities. In addition, secondary structure can be included in the HMM-HMM comparison, leading to another increase in sensitivity. We applied state-of-the-art HMM-HMM comparisons to the exocyst complex and detected structural similarity between all of the exocyst subunits.
We verified these structure predictions by identifying a structural domain in one of the exocyst subunits, Sec10p (YLR166C), from Saccharomyces cerevisiae
. Sec10p is one of the core subunits in the complex and has previously been shown to interact in vitro
with its partner exocyst subunits Sec6p, Exo70p and Sec15p 
. 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 
. 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 
. 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.