The COG and exocyst complexes are among the most intensively-studied MTCs, and partial structures of individual subunits demonstrate that they share homology on the tertiary structural level22–28
. Relatively little progress has, however, been made in characterizing these complexes in their assembled states. Previous quick-freeze/deep-etch EM analyses of the COG and exocyst complexes revealed evidence for multiple structural domains6,34
. Brief glutaraldehyde fixation in these studies, however, drastically altered the structural features of both complexes, leaving open the question of which samples the unfixed samples or the fixed samples – were more native-like in their properties. For COG, the fixed samples collapsed to form two globular lobes, leading to the prediction that one lobe contains the subunits essential in yeast (Cog1-4) while the other lobe contains the remaining subunits (Cog5-8)6
. While this division into functional domains has stood the test of time, the model of COG as composed of globular lobes is at odds with the results reported here. Instead, the essential (and presumably the remaining) COG subunits form long, thin legs in a decidedly spindly structure. This long-legged structure seems well-suited to a role in vesicle tethering at a distance. We note, however, that clathrin triskelia, which bear superficial resemblance to the Cog1-4 core complex, play an entirely different role in vesicle trafficking, serving as the protomers from which vesicle coats are assembled. Put differently, function does not necessarily follow form.
The four subunit N-termini play a fundamental role in complex assembly by bundling together along the proximal portion of one leg (). Consistent with this finding, deletion of 97 N-terminal residues from Cog2 is lethal24
. While our EM data could not further define the molecular details of the intersubunit interactions in the region in which all four subunits interdigitate, one attractive possibility is that each N-terminus contributes one (or more) α-helices to an extended helix bundle along this length of leg C. The diameter of such a bundle, were it to contain four helices, would be roughly the width of the leg as observed by EM. Furthermore, as first noted by Whyte and Munro1,8
, the N-terminal regions of COG subunits (and other CATCHR-family subunits) often contain regions predicted to form amphipathic α-helices; for yeast Cog2, the N-terminal region has been shown experimentally to adopt an ensemble of highly α-helical conformations24
. We therefore suggest that, in the Cog1-4 complex, the α-helical propensity of each subunit is further reinforced by quaternary interactions with the other subunits to form a bundle of four (or more) helices. Intriguingly, the core SNARE complex is a four-helix bundle of similar length35
. In the SNARE case, however, all four helices are parallel, whereas in our speculative model, the Cog1 and Cog2 helices would be antiparallel to the Cog3 and Cog4 helices. Finally, we note that a more general involvement of N-terminal regions in forming the quaternary interactions that stabilize CATCHR-family tethering complexes would explain why almost none of the known structures of isolated subunits include this region.
The distal ends of at least two of the three legs play important roles in COG complex function. The distal end of leg C, representing the C-terminus of Cog1, is important because it serves as a bridge to the non-essential subunits, Cog5-8. The distal end of leg B is assigned to the C-terminal portion of Cog4 based primarily on images of the core complex containing a C-terminal Cog4 truncation in Cog4 (). Also consistent with this assignment is the observation that the distal end of leg B is roughly congruent with the crystal structure of a 27-kDa C-terminal fragment of human Cog425
(). The crystal structure includes the site of an Arg 729 to Trp missense mutation that contributes to a congenital disorder of glycosylation36
and that results in severe glycosylation defects in HeLa cells without affecting the assembly or stability of the COG complex25
. Moreover, yeast harboring a C-terminally truncated version of Cog4 are inviable25
. These and other functional results25
strongly suggest that the distal end of leg B contains a binding site for an as-yet-unidentified trafficking factor. In the future, it will be interesting to investigate whether the C-terminus of Cog3, forming the distal tip of leg A, is likewise implicated in interactions with other components of the trafficking machinery. Finally, we note that human Cog2 is considerably larger than its yeast counterpart. Based on our model (), we propose that the human Cog1-4 complex may be “H”-shaped, with the fourth leg derived from Cog2. As the human Cog1 subunit is also larger, leg C may also be longer.
Other functionally important interaction sites map nearer the central junction of the Cog1-4 core complex. The N-terminal 150 residues of human Cog4 has been reported to harbor binding sites for the SNARE protein Syntaxin 5 and the Sec1/Munc18-family protein Sly116,37
. In the yeast COG core complex, this region of Cog4 resides along the proximal portion of leg C where all four subunits intertwine (). Overall, we conclude that the Cog1-4 complex likely engages other components of the trafficking machinery (including Cog5-8) using widely-distributed sites located at the tips of at least two legs and within the central core.
The structural organization of the COG complex presented here invites comparison with the Dsl1 complex, whose structure was reported recently22,23
. At the tertiary structure level, there are significant similarities between some COG and Dsl1 complex subunits22–25
. At the quaternary structure level, the interactions that stabilize each complex involve regions at or near the termini of the individual subunits. All four of the essential COG subunits interact by means of their N-terminal segments; likewise, two of the three Dsl1 complex subunits (Tip20 and Dsl1) interact via the anti-parallel pairing of N-terminal helices23
. A second similarity between the COG and Dsl1 complexes is that each of them contains a single subunit (Cog1 and Dsl1, respectively) that functions as a bridge by interacting at (or near) each end with different subunits. Finally, like the COG complex, the Dsl1 complex contains spatially distant sites important for interaction with other components of the trafficking machinery22,23
. Nonetheless, despite these similarities, the overall architecture of the Dsl1 complex and the COG core complex are quite different. Further study will be needed to determine the extent to which these complexes, as well as the exocyst and GARP complexes, operate according to common mechanistic principles.