Conservation of Helical Bundle Structure between the Exocyst Subunits

Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2009; 4(2):e4443. DOI: 10.1371/journal.pone.0004443
Source: PubMed


The 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.
Using 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% alpha-helicity, in agreement with our predictions, and capable of interacting with several known Sec10p binding partners.
Although 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.

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    • "Sec5; (b) Duplication of grapevine Sec6; (c) Origin of two Sec3 clades (monocot1 and monocot2), and of the Sec15 clades A1 and A2; (d) Origin of Sec15 clades A and B; (e) Origin of Exo84 clades A/B and C/CX; (f) Separation of the Exo84 clades A and B, as well as C and CX, the later subsequently lost in some descendants; (g) Amplification of dicot Exo70 clade H; (h) Amplification of the monocot Exo70 clade FX. genes appear to have two closely related paralogs, albeit this species is believed to be one of the few plants without a recent history of whole-genome duplications (Jiao et al., 2011). We have found that a subgroup of exocyst subunits, corresponding to the previously proposed core of the complex (Munson and Novick, 2006; Croteau et al., 2009), underwent little or no amplification in the vascular plants, though even these subunits have amplified to a some extent in non-seed plants. These low copy subunits, in particular Sec6, Sec8, and Sec10, but to a somewhat lesser extent also Sec5 and Sec3, exhibit evolutionary trees that are not only topologically similar but also obviously correlated in terms of branch length, which is consistent with co-evolution driven by the requirement of maintaining mutual compatibility of closely interacting complex subunits (Juan et al., 2008; Lovell and Robertson, 2010). "
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    ABSTRACT: Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits - Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 - were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single "Exocyst complex" - instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.
    Frontiers in Plant Science 07/2012; 3:159. DOI:10.3389/fpls.2012.00159 · 3.95 Impact Factor
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    • "Recombinant Sec6 (1–805; Sivaram et al., 2005), Exo70 (63–623; Dong et al., 2005), Exo84CT (523–753; Dong et al., 2005), Sec10 (145–827; Croteau et al., 2009), Sec8 (Sivaram et al., 2006), and the cytoplasmic regions of the SNARE proteins Sso1, Snc2, and Sec9CT we propose that Sec6–Sec9 holds Sec9 in an inactive state (perhaps an assembly intermediate) at sites of secretion, where Sso1 becomes activated, to prevent premature or inappropriate SNARE assembly and vesicle fusion. The small amount of Sec6 that is Sec9 bound (Figure 4) is consistent with the low abundance of Sec9 localized to sites of exocytosis. "
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    ABSTRACT: Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.
    Molecular biology of the cell 11/2011; 23(2):337-46. DOI:10.1091/mbc.E11-08-0670 · 4.47 Impact Factor
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    • "The related electron microscopic structural studies on living plant materials were mostly performed between the 1960s to early 1980s (Palade and Bruns 1968, Lucy 1970, Takeo et al. 1973, Heuser et al. 1974, 1979, Chandler and Heuser 1980). Recent progresses are made mainly through applying molecular methods and other new technologies (Cho et al. 2002, 2004, Yang and Huang 2002, Jahn et al. 2003, Jeremic et al. 2003, Jahn 2004, Jena 2004, 2005a, 2005b, 2006, 2010, Kelly et al. 2004, Chernomordik and Kozlov 2005, Jahn and Scheller 2006, Siksou et al. 2007, Croteau et al. 2009, Songer and Munson 2009, Lee et al. 2010, Zhang et al. 2010). Chandler and Heuser (1980) provided details about membrane fusion through TEM observation on samples prepared by quick freezing. "
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    ABSTRACT: Studies on membrane fusion in living cells indicate that initiation of membrane fusion is a transient and hard to capture process. Despite previous research, membrane behaviour at this point is still poorly understood. Recent palaeobotanical research has revealed snapshots of membrane fusion in a 15-million-year-old fossil pinaceous cone. To reveal the membrane behaviour during the fusion, we conducted more observations on the same fossil material. Several discernible steps of membrane fusion have been fixed naturally and observed in the fossil material. This observation provides transmission electron microscope (TEM) images of the transient intermediate stage and clearly shows the relationship between membranes. Observing such a transient phenomenon in fossil material implies that the fixing was most likely accomplished quickly by a natural process. The mechanism behind this phenomenon is clearly worthy of further enquiry.
    Molecular Membrane Biology 02/2011; 28(2):115-22. DOI:10.3109/09687688.2010.536169 · 1.69 Impact Factor
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