Julien Béthune

Membrane curvature induced by Arf1-GTP is essential for vesicle formation.

The GTPase Arf1 is considered as a molecular switch that regulates binding and release of coat proteins that polymerize on membranes to form transport vesicles. Here, we show that Arf1-GTP induces positive membrane curvature and find that the small GTPase can dimerize dependent on GTP. Investigating a possible link between Arf dimerization and curvature formation, we isolated an Arf1 mutant that cannot dimerize. Although it was capable of exerting the classical role of Arf1 as a coat receptor, it could not mediate the formation of COPI vesicles from Golgi-membranes and was lethal when expressed in yeast. Strikingly, this mutant was not able to deform membranes, suggesting that GTP-induced dimerization of Arf1 is a critical step inducing membrane curvature during the formation of coated vesicles.

COPI-mediated Transport

COPI-coated vesicles are protein and liquid carriers that mediate transport within the early secretory pathway. In this Topical Review, we present their main protein components and discuss current models for cargo sorting. Finally, we describe the striking similarities that exist between the COPI system and the two other characterized types of vesicular carriers: COPII- and clathrin-coated vesicles.

Coatomer, the Coat Protein of COPI Transport Vesicles, Discriminates Endoplasmic Reticulum Residents from p24 Proteins

ABSTRACT In the formation of COPI vesicles, interactions take place between the coat protein coatomer and membrane proteins: either cargo proteins for retrieval to the endoplasmic reticulum (ER) or proteins that cycle between the ER and the Golgi. While the binding sites on coatomer for ER residents have been characterized, how cycling proteins bind to the COPI coat is still not clear. In order to understand at a molecular level the mechanism of uptake of such proteins, we have investigated the binding to coatomer of p24 proteins as examples of cycling proteins as well as that of ER-resident cargos. The p24 proteins required dimerization to interact with coatomer at two independent binding sites in γ-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than γ-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.

Multiple and stepwise interactions between coatomer and ADP-ribosylation factor-1 (Arf1)-GTP.

The small GTPase ADP‐ribosylation factor‐1 (Arf1) plays a key role in the formation of coat protein I (COP I)‐coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1’s GDP is exchanged for GTP. Arf1‐GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer β and γ‐COP subunits through its switch I region, and with ɛ‐COP. Here, we mapped the interaction of the Arf1‐GTP switch I region to the trunk domains of β and γ‐COP. Site‐directed photolabeling at position 167 in the C‐terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of δ‐COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane‐bound Arf1‐GTP. These interactions are located within the core, adaptor‐like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.

A Conformational Change in the α-subunit of Coatomer Induced by Ligand Binding to γ-COP Revealed by Single-pair FRET

Formation of transport vesicles involves polymerization of cytoplasmic coat proteins (COP). In COPI vesicle biogenesis, the heptameric complex coatomer is recruited to donor membranes by the interaction of multiple coatomer subunits with the budding machinery. Specific binding to the trunk domain of γ‐COP by the Golgi membrane protein p23 induces a conformational change that causes polymerization of the complex. Using single‐pair fluorescence resonance energy transfer, we find that this conformational change takes place in individual coatomer complexes, independent of each other, and that the conformational rearrangement induced in γ‐COP is transmitted within the complex to its α‐subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.

Conformational changes of coat proteins during vesicle formation

In coated vesicle formation, coat protein recruitment needs to be spatially and temporally controlled. The coating process involves conformational changes of the coat protein complexes that activate them for interaction with cargo or machinery components and coat polymerization. Here we discuss mechanisms that have emerged recently from studies of the clathrin adaptor and the COPI systems.

Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes

Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein–protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.

4EHP-independent repression of endogenous mRNAs by the RNA-binding protein GIGYF2

Abstract Initially identified as a factor involved in tyrosine kinase receptor signaling, Grb10-interacting GYF protein 2 (GIGYF2) has later been shown to interact with the 5′ cap-binding protein 4EHP as part of a translation repression complex, and to mediate post-transcriptional repression of tethered reporter mRNAs. A current model proposes that GIGYF2 is indirectly recruited to mRNAs by specific RNA-binding proteins (RBPs) leading to translation repression through its association with 4EHP. Accordingly, we recently observed that GIGYF2 also interacts with the miRNA-induced silencing complex and probably modulates its translation repression activity. Here we have further investigated how GIGYF2 represses mRNA function. In a tethering reporter assay, we identify three independent domains of GIGYF2 with repressive activity. In this assay, GIGYF2-mediated repression is independent of 4EHP but largely dependent on the CCR4/NOT complex that GIGYF2 recruits through multiple interfaces. Importantly, we show that GIGYF2 is an RBP and identify for the first time endogenous mRNA targets that recapitulate 4EHP-independent repression. Altogether, we propose that GIGYF2 has two distinct mechanisms of repression: one depends on 4EHP binding and mainly affects translation; the other is 4EHP-independent and involves the CCR4/NOT complex and its deadenylation activity.

Assembly of COPI and COPII Vesicular Coat Proteins on Membranes

In eukaryotes, distinct transport vesicles functionally connect various intracellular compartments. These carriers mediate transport of membranes for the biogenesis and maintenance of organelles, secretion of cargo proteins and peptides, and uptake of cargo into the cell. Transport vesicles have distinct protein coats that assemble on a donor membrane where they can select cargo and curve the membrane to form a bud. A multitude of structural elements of coat proteins have been solved by X-ray crystallography. More recently, the architectures of the COPI and COPII coats were elucidated in context with their membrane by cryo-electron tomography. Here, we describe insights gained from the structures of these two coat lattices and discuss the resulting functional implications.

Split-BioID — Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.