Vesicles: Looking inside the cell
Advances in imaging techniques have shed new light on the structure of vesicles formed by COPI protein complexes.
- University Medical Center Göttingen, Germany
Vesicles perform a wide range of functions within cells, such as the transport of proteins and lipids between the different parts of a cell. Each vesicle is coated with a protein complex, and understanding the structure and function of these complexes is a central challenge in cell biology. Two of these complexes – COPII and clathrin – are relatively well understood, but the third, which is called COPI, is not.
Vesicles coated with COPI are responsible for transport from the Golgi to the endoplasmic reticulum, and also for transport between the cisternae within the Golgi (Beck et al., 2009). The COPI complex contains seven subunits that are recruited en bloc to the vesicle membrane (Hara-Kuge et al., 1994). However, the intricate couplings between these subunits have long hindered efforts to determine the molecular architecture of the COPI coats.
The basic model of COPI vesicle formation begins with the recruitment of a GTPase called Arf1 to a membrane. Arf1 is then loaded with GTP, thereby activating it. COPI complexes then interact with the GTP-loaded Arf1 and cargo proteins to form the vesicle, which can then detach from the membrane and transport the cargo proteins to their destination. Finally, the coat disassembles in a process that is called un-coating: three GTPase activating proteins (ArfGAP1/2/3) play an important role in this process by stimulating the hydrolysis of the GTP and, therefore, deactivating Arf1 (Lanoix et al., 1999; Pepperkok et al., 2000; Weimer et al., 2008). Subsequent studies of this un-coating process suggested that Arf1 and COPI disengage from membranes independently, in a non-cooperative manner (Presley et al., 2002; Yang et al., 2002). These and other results led to a model in which the individual COPI complexes were held in place by lateral linkages within the coat and by interactions with the underlying cargo.
These studies were built upon in a series of in-vitro reconstitution experiments undertaken by a collaboration between the groups of Felix Wieland (University of Heidelberg) and John Briggs (European Molecular Biology Laboratory and the MRC Laboratory of Molecular Biology). The in-vitro reconstitution approach involves combining purified proteins and liposomes (a type of vesicle) in a test tube to generate COPI-coated vesicles, thus allowing researchers to study the precise sequence of events that take place during vesicle formation. Wieland, Briggs and co-workers exploited significant advances in two techniques – cryo-electron tomography and subtomogram averaging – to determine the molecular structure of the COPI coat (Dodonova et al., 2017; Dodonova et al., 2015; Faini et al., 2012).
However, the process of vesicle formation in these seminal experiments differed from the process in real cells in a number of ways: the vesicles did not contain any cargo, and the Arf1 was frozen in its activated state because the researchers used a variant of GTP called GTPγS that cannot be hydrolysed. This meant that several questions remained unanswered: Does early GTP hydrolysis contribute to a pronounced conformational change of the coat (such as the exposure of cargo-recognition sites)? Are the linkages within the coat rearranged to accommodate cargo? How does Arf1 disengage from COPI without perturbing the pre-existing lattice? And how does the cage adapt structurally to the loss of Arf1?
The rational next step would have involved using GTP (rather than GTPγS), the three ArfGAPs and cargo proteins in in-vitro reconstitution experiments, which would have been very arduous. However, in an unexpected development reported in eLife, Briggs, Benjamin Engel and Wolfgang Baumeister (both of the Max Planck Institute of Biochemistry) and co-workers – including Yury Bykov as first author – describe the native structure of COPI-coated vesicles in a species of green algae called Chlamydomonas reinhardtii (Bykov et al., 2017). This quantum leap was made possible through a combination of cryo-focused ion-beam milling (a technique that involves vitrifying cells and then using a focused ion beam to slice off ultrathin sections) and further improvements in cryo-electron tomography and subtomogram averaging. Moreover, this approach meant that the researchers were able to study the structure of the coat in the presence of active Arf1, the relevant ArfGAPs and the vesicle's cargo.
Building on their previous systematic characterization of the morphology of the Golgi in C. reinhardtii (Engel et al., 2015a; Engel et al., 2015b), and what is known about the structures of COPI, COPII and clathrin, Bykov et al. were able to identify the three different types of coated vesicles in the vicinity of the cisternae within the Golgi. The researchers were also able to determine the in-situ structure of the coat without relying on existing structural models, and found that it was remarkably similar to the structure that emerged from the in-vitro reconstitution experiments. This finding is significant for the field because it strongly suggests that the existing structural model is physiologically relevant.
Moreover, an additional density was observed inside the vesicles in C. reinhardtii that was not visible in the in-vitro experiments. This density is likely to correspond to cargo, and the fact that it was observed under the β-propeller of the β’-subunit, but not beneath the β-propeller of the α-subunit (which is structurally similar), suggests that this cargo is recognized by a specific subunit of COPI. It is also striking that this density is seen in many vesicles adjoining both the cis and medial cisternae.
Remarkably, the relative proportions of Arf1 and COPI were the same for most vesicles, irrespective of the intactness of the coat: this suggests that, upon hydrolysis, the constituents of the coat are all released at the same time to allow the vesicle to fuse with an acceptor compartment, and argues against a step-wise dissociation of the coat from the vesicle.
In addition to demonstrating the ability of in-situ experiments to distinguish between models based on in-vitro experiments, the work of Bykov et al. highlights the ability of new techniques to allow us to see things – such as the density under the β-propeller of the β’-subunit – that we have missed so far. We are keen to get a glimpse of COPII and clathrin in situ too.
References
-
The COPI system: molecular mechanisms and functionFEBS Letters 583:2701–2709.https://doi.org/10.1016/j.febslet.2009.07.032
-
En bloc incorporation of coatomer subunits during the assembly of COP-coated vesiclesJournal of Cell Biology 124:883–892.
-
COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargoJournal of Cell Science 113 (Pt 1):135–144.
-
Differential roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI traffickingJournal of Cell Biology 183:725–735.https://doi.org/10.1083/jcb.200806140
-
ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coatJournal of Cell Biology 159:69–78.https://doi.org/10.1083/jcb.200206015
Article and author information
Author details
Blanche Schwappach
Publication history
- Version of Record published: December 5, 2017 (version 1)
Copyright
© 2017, Arakel et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,169
- Page views
-
- 257
- Downloads
-
- 1
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Vesicles: Looking inside the cell
eLife 6:e33650.
https://doi.org/10.7554/eLife.33650
Further reading
-
- Structural Biology and Molecular Biophysics
Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules’ diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.
-
- Structural Biology and Molecular Biophysics
Volume-regulated anion channels (VRACs) mediate volume regulatory Cl- and organic solute efflux from vertebrate cells. VRACs are heteromeric assemblies of LRRC8A-E proteins with unknown stoichiometries. Homomeric LRRC8A and LRRC8D channels have a small pore, hexameric structure. However, these channels are either non-functional or exhibit abnormal regulation and pharmacology, limiting their utility for structure-function analyses. We circumvented these limitations by developing novel homomeric LRRC8 chimeric channels with functional properties consistent with those of native VRAC/LRRC8 channels. We demonstrate here that the LRRC8C-LRRC8A(IL125) chimera comprising LRRC8C and 25 amino acids unique to the first intracellular loop (IL1) of LRRC8A has a heptameric structure like that of homologous pannexin channels. Unlike homomeric LRRC8A and LRRC8D channels, heptameric LRRC8C-LRRC8A(IL125) channels have a large-diameter pore similar to that estimated for native VRACs, exhibit normal DCPIB pharmacology, and have higher permeability to large organic anions. Lipid-like densities are located between LRRC8C-LRRC8A(IL125) subunits and occlude the channel pore. Our findings provide new insights into VRAC/LRRC8 channel structure and suggest that lipids may play important roles in channel gating and regulation.
