Department of Biology, University of Pennsylvania, Philadelphia, PA, USA.
Ministry of Education Key Laboratory of Protein Sciences, Tsinghua University,
Beijing Advanced Innovation Center for Structural Biology, Tsinghua University, Beijing, China.
School of Life Sciences, Tsinghua
University, Beijing, China.
Tsinghua-Peking Joint Center for Life Sciences, Beijing, China.
National Institute of Biological Sciences, Beijing, China. Present
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. Kunrong Mei and Yan Li
contributed equally to this work. *e-mail: firstname.lastname@example.org; email@example.com
esicular trafficking in eukaryotic cells is mediated by an
elaborate network of molecular interactions that ensure the
orderly transport, docking and fusion of secretory vesicles
to their cognate target membrane. The initial contacts between the
secretory vesicles and their target membrane are mediated by the
tether family of proteins
. The multisubunit tethering complexes
(MTCs) capture the vesicles to their specific target membranes
before SNARE-mediated fusion at various stages of vesicular traf
. Elucidating the structure and assembly of the MTCs is
essential to the understanding of the mechanisms of vesicle tether
ing and fusion.
The exocyst, first identified in the budding yeast S. cerevisiae,
consists of the proteins Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70
and Exo84 (refs
). The exocyst mediates the tethering of post-
Golgi secretory vesicles to the plasma membrane and promotes the
assembly of the SNARE complex for membrane fusion
from exocytosis, the exocyst also plays pivotal roles in many cellular
processes such as cell polarization, primary ciliogenesis, cytokine
sis, pathogen invasion, tumorigenesis and metastasis
crystallography studies have revealed important structural features
of a few exocyst subunits; these regions are mostly helical-bundle
. Quick-freeze deep-etch EM and negative-staining EM
have shown that the exocyst has an elongated shape consisting of
packed long rods
. The rod structure seems to be a shared fea-
ture for complexes associated with tethering containing helical rods
(CATCHR) proteins such as the conserved oligomeric Golgi (COG)
and Golgi-associated retrograde protein (GARP) complexes
Recently, a light microscopy approach, based on fluorescent protein
tagging, provided new information on the exocyst complex in cells
A topology regarding the connectivity of the exocyst subunits has
also been depicted using biochemical analyses and negative-stain
. However, the structure model and mechanism of exocyst
assembly remain unknown.
Using single-particle cryo-EM, we have solved the structure of
the fully assembled yeast exocyst complex at an average resolution
of 4.4 Å. The cryo-EM, together with chemical cross-linking mass
spectrometry (CXMS) and cell biological assays, provides insights
into the hierarchical assembly of the exocyst complex and mecha
nism of vesicle tethering.
Structure determination of the exocyst complex. We tagged the
chromosomal copy of individual exocyst subunits with TAP or ProA
and purified the exocyst complex from yeast cell lysates using affin
ity chromatography and size-exclusion chromatography (Fig. 1a,b).
The purification yielded a monodispersed intact exocyst complex
comprising all of the eight subunits at equal stoichiometry (Fig. 1b).
We next performed single-particle cryo-EM analysis of the complex
and reconstituted the 3D structure of the intact complex at an aver
age resolution of 4.4 Å (Fig. 1c,d and Supplementary Figs. 1 and 2).
The holo-exocyst complex consists of long curved rods that pack
against each other to form a hollow architecture measuring 320 Å
long and 130 Å wide (Fig. 1d). The central body appears as a double-
layer oval disk with a thickness of 60 Å. The front layer nestles into
the concave side of the back layer, forming a prolate cave between
the two layers. From the top of the front layer, a neck-like struc
ture stretches out, with two arms extending in opposite directions.
One arm protrudes to the right edge of the front layer (henceforth
termed arm I), and the other extends backward over the back layer
(termed arm II). Stretching out from the bottom of the back layer
is a tail-like structure (termed tail). Whereas the central portion of
the reconstruction clearly shows bundles of α -helices with appar
ent helical pitches, the arm I, arm II and tail regions are less well
resolved (Fig. 1d and Supplementary Fig. 2e), thus suggesting flex
ibility in these regions.
The assignment and structure of the exocyst subunits. The 4.4-Å
resolution does not allow clear resolution of the side chains. To
gain better structural information on the exocyst, we performed
extensive intra- and intermolecular CXMS analyses. The com
bined approach allowed us to build a model of the exocyst complex
consisting of the near full-length Sec5, Sec6, Sec8, Sec10, Sec15,
Cryo-EM structure of the exocyst complex
, Yan Li
, Shaoxiao Wang
, Guangcan Shao
, Jia Wang
, Yuehe Ding
, Peng Yue
, Jun-Jie Liu
, Xinquan Wang
, Meng-Qiu Dong
* and Wei Guo
The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory ves-
icles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytoki-
nesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the
Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst
and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis
suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a
mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
NATURE STRUCTURAL & MOLECULAR BIOLOGY | VOL 25 | FEBRUARY 2018 | 139–146 | www.nature.com/nsmb
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