Abstract
BackgroundThe exocyst is a large hetero-octomeric protein complex required for regulating the targeting and fusion of secretory vesicles to the plasma membrane in eukaryotic cells. Although the sequence identity between the eight different exocyst subunits is less than 10%, structures of domains of four of the subunits revealed a similar helical bundle topology. Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies.Methodology/Principal FindingsUsing advanced hidden Markov models combined with secondary structure predictions, we detect significant sequence similarity between each of the exocyst subunits, indicating that they all contain helical bundle structures. We corroborate these remote homology predictions by identifying and purifying a predicted domain of yeast Sec10p, a previously insoluble exocyst subunit. This domain is soluble and folded with approximately 60% α-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners.Conclusions/SignificanceAlthough all eight of the exocyst subunits had been suggested to be composed of similar helical bundles, this has now been validated by our hidden Markov model structure predictions. In addition, these predictions identified protein domains within the exocyst subunits, resulting in creation and characterization of a soluble, folded domain of Sec10p.
Highlights
The exocyst is a large, eight protein complex localized to sites of polarized secretion that is required for exocytosis and cytokinesis in eukaryotes ([1,2,3]; and references therein)
Each of the exocyst subunits is predicted to be a-helical. They possess less than 10% sequence identity with each other, limited sequence similarity has been detected using PSI-BLAST analyses [7,11]
The exocyst complex is composed of eight large proteins that are predicted to be predominantly helical by secondary structure predictions (Figure 1B)
Summary
The exocyst is a large, eight protein complex localized to sites of polarized secretion that is required for exocytosis and cytokinesis in eukaryotes ([1,2,3]; and references therein). Each of the exocyst subunits is predicted to be a-helical They possess less than 10% sequence identity with each other, limited sequence similarity has been detected using PSI-BLAST analyses [7,11]. They show little similarity to other proteins or domains, except for short regions of predicted coiled coils [1,7]. Several recent crystal structures of domains from individual subunits have been determined: nearly full-length yeast and human Exo70 [12,13,14], and the C-terminal domains of yeast Exo84p [12], yeast Sec6p [15] and Drosophila Sec15 [16] They show similar structures containing multiple helical bundles, yielding an overall similar shape (Figure 1A). Characterization of several of these subunits has been hindered by lack of soluble protein for biochemical and structural studies
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