Journal of Molecular Biology
The Crystal Structure of Mouse Exo70 Reveals Unique Features of the Mammalian Exocyst
Introduction
Intracellular vesicle delivery depends upon a series of protein complexes working in tandem to transport, tether, and fuse vesicles to specific target membranes. Tethering complexes are thought to mediate the SNARE-dependent fusion of these vesicles to the target membrane at specific sites. In exocytosis, exocytic vesicles are transported from the Golgi apparatus along the cytoskeleton to specific fusion sites on the plasma membrane (PM) by the unconventional myosin Myo2 in yeast1., 2. and others in mammals. The exocyst is a multi-subunit tethering complex involved in exocytosis and receptor recycling.3., 4., 5., 6. It has been identified in most eukaryotes7 and is composed of eight protein subunits: Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84.3., 8., 9., 10. The exocyst is the first contact between the vesicle and the PM in exocytosis and is known to regulate exocytosis through interactions with multiple small GTPases in a GTP-dependent manner, including Rho1,11 Rho3,12 Cdc42,13 and Sec414 in yeasts; and RalA,15., 16., 17., 18. TC10,19 Arf6,5 and Rab1120 in mammals. Disruption of the exocyst function does not affect the ability of exocytic vesicles to reach the PM, but it does affect the ability of these vesicles to fuse with the PM.21., 22.
Exo70 is the 70 kDa subunit of the exocyst and its primary sequence is conserved only weakly among Exo70 orthologs (16% identity and 35% similarity between Mus musculus and Saccharomyces cerevisiae). Within the exocyst, Exo70 is known to interact with Sec6,23., 24. Sec8,23., 25. and Sec10;23., 25. and with Sec15 and Exo84 in mammals,24 and Sec5 in yeast.14 More recently, Arpc1, a subunit of the Arp2/3 complex, has been shown to interact with Exo70, and this interaction is important for actin network reorganization at the PM in both yeast and mammals.26 Exo70 also mediates interactions between the exocyst and small GTPases, which appears to have a role in the regulation and assembly of the exocyst. Interaction with activated TC10 regulates the presence of exocytic sites at lipid rafts in insulin-stimulated glucose transport in mammals.19., 27. In yeast, interaction with activated Rho3 controls the fusion of exocytic vesicles with the PM,12., 28. and may be important in demarcation of exocytic sites.29 Despite some overlapping functions, however, interaction with these small GTPases occurs at two separate sites on Exo70,19., 23. suggesting that the functional organization of Exo70 may not be entirely conserved. While the recently reported structures of S. cerevisiae Exo70 (ScExo70)23., 30. have been used as a general model to understand the structure and function relationship of all Exo70 proteins, it is essential to obtain high-resolution structural information of mammalian Exo70 in order to address questions specific to mammalian exocytosis.
Here, we report the crystal structure of M. musculus Exo70 (MmExo70) at 2.25 Å resolution. Comparison with the structure of ScExo70 reveals that while the overall fold of the protein is conserved, there is significant structural reorganization within the molecule that results in the reorientation of the C-terminal domain relative to the rest of the molecule. In addition, loops connecting secondary structural elements show significant differences in length and conformation. This, combined with a general lack of primary sequence conservation, results in the alteration of the molecule's surface properties. The observed structural similarity and differences are consistent with the function of Exo70 as an exocyst subunit that interacts with different sets of effector molecules in different organisms. The structure of MmExo70 is the first structure of a mammalian exocyst subunit and makes Exo70 the first exocyst subunit with structural information available in more than one organism.
Section snippets
Structure determination
To obtain diffraction-quality crystals of MmExo70, purified full-length protein was subjected to limited proteolysis by subtilisin, which identified a major fragment containing a deletion of the N-terminal 84 residues of the molecule. This fragment (residues 85–653) was crystallized in the P3221 space group with unit cell dimensions of a = b = 61.5 Å, c = 294.7 Å and one molecule in the asymmetric unit. The structure was determined by the multi-wavelength anomalous diffraction (MAD) method using
MmExo70 expression and purification
The MmExo70Δ84 gene (coding for residues 85–653) was amplified from M. musculus cDNA (provided by A. Saltiel) by PCR and subcloned into the pSJ7 vector, a derivative of pET43a (Novagen). The native protein was expressed in Escherichia coli BL21(DE3) strain containing the expression plasmid at 16 °C in LB medium, and the l-selenomethionine variant protein was expressed in E. coli strain B834(DE3) containing the expression plasmid at 16 °C in minimal medium containing l-selenomethionine. Cells
Acknowledgements
We thank the staff of the Center for Structural Biology at the University of Michigan for maintaining the X-ray facility; the staff at the APS GM/CA-CAT beamline in the Argonne National Laboratory for access and help with data collection; and Alan R. Saltiel, Janet L. Smith, and John Tesmer for critical reading of the manuscript. B.M. was a trainee in the Molecular Mechanisms in Microbial Pathogenesis training program at the University of Michigan Medical School. This work was supported, in
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