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
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
-
- 2,463
- views
-
- 260
- downloads
-
- 1
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
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
-
- Biochemistry and Chemical Biology
- Structural Biology and Molecular Biophysics
Lipopolysaccharides (LPS) confer resistance against harsh conditions, including antibiotics, in Gram-negative bacteria. The lipopolysaccharide transport (Lpt) complex, consisting of seven proteins (A-G), exports LPS across the cellular envelope. LptB2FG forms an ATP-binding cassette transporter that transfers LPS to LptC. How LptB2FG couples ATP binding and hydrolysis with LPS transport to LptC remains unclear. We observed the conformational heterogeneity of LptB2FG and LptB2FGC in micelles and/or proteoliposomes using pulsed dipolar electron spin resonance spectroscopy. Additionally, we monitored LPS binding and release using laser-induced liquid bead ion desorption mass spectrometry. The β-jellyroll domain of LptF stably interacts with the LptG and LptC β-jellyrolls in both the apo and vanadate-trapped states. ATP binding at the cytoplasmic side is allosterically coupled to the selective opening of the periplasmic LptF β-jellyroll domain. In LptB2FG, ATP binding closes the nucleotide binding domains, causing a collapse of the first lateral gate as observed in structures. However, the second lateral gate, which forms the putative entry site for LPS, exhibits a heterogeneous conformation. LptC binding limits the flexibility of this gate to two conformations, likely representing the helix of LptC as either released from or inserted into the transmembrane domains. Our results reveal the regulation of the LPS entry gate through the dynamic behavior of the LptC transmembrane helix, while its β-jellyroll domain is anchored in the periplasm. This, combined with long-range ATP-dependent allosteric gating of the LptF β-jellyroll domain, may ensure efficient and unidirectional transport of LPS across the periplasm.
-
- Structural Biology and Molecular Biophysics
Polycystin-1 (PC1) is the protein product of the PKD1 gene whose mutation causes autosomal dominant Polycystic Kidney Disease (ADPKD). PC1 is an atypical G protein-coupled receptor (GPCR) with an autocatalytic GAIN domain that cleaves PC1 into extracellular N-terminal and membrane-embedded C-terminal (CTF) fragments. Recently, activation of PC1 CTF signaling was shown to be regulated by a stalk tethered agonist (TA), resembling the mechanism observed for adhesion GPCRs. Here, synthetic peptides of the first 9- (p9), 17- (p17), and 21-residues (p21) of the PC1 stalk TA were shown to re-activate signaling by a stalkless CTF mutant in human cell culture assays. Novel Peptide Gaussian accelerated molecular dynamics (Pep-GaMD) simulations elucidated binding conformations of p9, p17, and p21 and revealed multiple specific binding regions to the stalkless CTF. Peptide agonists binding to the TOP domain of PC1 induced close TOP-putative pore loop interactions, a characteristic feature of stalk TA-mediated PC1 CTF activation. Additional sequence coevolution analyses showed the peptide binding regions were consistent with covarying residue pairs identified between the TOP domain and the stalk TA. These insights into the structural dynamic mechanism of PC1 activation by TA peptide agonists provide an in-depth understanding that will facilitate the development of therapeutics targeting PC1 for ADPKD treatment.
