Engineering of ultraID, a compact and hyperactive enzyme for proximity-dependent biotinylation in living cells.

Proximity-dependent biotinylation (PDB) combined with mass spectrometry analysis has established itself as a key technology to study protein-protein interactions in living cells. A widespread approach, BioID, uses an abortive variant of the E. coli BirA biotin protein ligase, a quite bulky enzyme with slow labeling kinetics. To improve PDB versatility and speed, various enzymes have been developed by different approaches. Here we present a small-size engineered enzyme: ultraID. We show its practical use to probe the interactome of Argonaute-2 after a 10 min labeling pulse and expression at physiological levels. Moreover, using ultraID, we provide a membrane-associated interactome of coatomer, the coat protein complex of COPI vesicles. To date, ultraID is the smallest and most efficient biotin ligase available for PDB and offers the possibility of investigating interactomes at a high temporal resolution.

Context-Specific and Proximity-Dependent Labeling for the Proteomic Analysis of Spatiotemporally Defined Protein Complexes with Split-BioID.

Proximity-dependent labeling techniques such as BioID and APEX2 allow the biotinylation of proteins proximal to a protein of interest in living cells. Following streptavidin pulldown and mass spectrometry analysis, this enables the identification of native protein-protein interactions. Here we describe split-BioID, a protein-fragment complementation assay that increases the resolution of BioID. Using this technique, context-specific protein complexes can be resolved.

A paralog-specific role of COPI vesicles in the neuronal differentiation of mouse pluripotent cells.

Coat protein complex I (COPI)-coated vesicles mediate membrane trafficking between Golgi cisternae as well as retrieval of proteins from the Golgi to the endoplasmic reticulum. There are several flavors of the COPI coat defined by paralogous subunits of the protein complex coatomer. However, whether paralogous COPI proteins have specific functions is currently unknown. Here, we show that the paralogous coatomer subunits γ1-COP and γ2-COP are differentially expressed during the neuronal differentiation of mouse pluripotent cells. Moreover, through a combination of genome editing experiments, we demonstrate that whereas γ-COP paralogs are largely functionally redundant, γ1-COP specifically promotes neurite outgrowth. Our work stresses a role of the COPI pathway in neuronal polarization and provides evidence for distinct functions for coatomer paralogous subunits in this process.

Protease-resistant streptavidin for interaction proteomics.

Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.

Proteomic Profiling of Mammalian COPII and COPI Vesicles.

Intracellular transport and homeostasis of the endomembrane system in eukaryotic cells depend on the formation and fusion of vesicular carriers. Coat protein complex (COP) II vesicles export newly synthesized secretory proteins from the endoplasmic reticulum (ER), whereas COPI vesicles facilitate traffic from the Golgi to the ER and intra-Golgi transport. Mammalian cells express various isoforms of COPII and COPI coat proteins. To investigate the roles of coat protein paralogs, we have combined in vitro vesicle reconstitution from semi-intact cells with SILAC-based mass spectrometric analysis. Here, we describe the core proteomes of mammalian COPII and COPI vesicles. Whereas the compositions of COPII vesicles reconstituted with various isoforms of the cargo-binding subunit Sec24 differ depending on the paralog used, all of the isoforms of the COPI coat produce COPI-coated vesicles with strikingly similar protein compositions.

Membrane-Associated RNA-Binding Proteins Orchestrate Organelle-Coupled Translation.

Proteins are positioned and act at defined subcellular locations. This is particularly important in eukaryotic cells that deliver proteins to membrane-bound organelles such as the endoplasmic reticulum (ER), mitochondria, or endosomes. It is axiomatic that organelle targeting depends mainly on polypeptide signals. However, recent results demonstrate that targeting elements within the encoding transcripts are essential for efficient protein localisation. Key readers of these elements are membrane-associated RNA-binding proteins (memRBPs) that orchestrate organelle-coupled translation. The translation products then either cross the membrane for organelle entry or hitchhike on organelle surfaces for complex assembly and co-transport. Understanding the interaction of protein- and RNA-based targeting signals is essential to decipher the molecular basis for mutant phenotypes in disease.

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.

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

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 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.

Kinetic analysis reveals successive steps leading to miRNA-mediated silencing in mammalian cells.

MicroRNAs (miRNAs) regulate most cellular functions, acting by posttranscriptionally repressing numerous eukaryotic mRNAs. They lead to translational repression, deadenylation and degradation of their target mRNAs. Yet, the relative contributions of these effects are controversial and little is known about the sequence of events occurring during the miRNA-induced response. Using stable human cell lines expressing inducible reporters, we found that translational repression is the dominant effect of miRNAs on newly synthesized targets. This step is followed by mRNA deadenylation and decay, which is the dominant effect at steady state. Our findings have important implications for understanding the mechanism of silencing and reconcile seemingly contradictory data.

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.

A conformational change in the alpha-subunit of coatomer induced by ligand binding to gamma-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 gamma-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 gamma-COP is transmitted within the complex to its alpha-subunit. We suggest that capture of membrane protein machinery triggers cage formation in the COPI system.

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 beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-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 delta-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.

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.

Coatomer, the coat protein of COPI transport vesicles, discriminates endoplasmic reticulum residents from p24 proteins.

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 gamma-COP. In contrast, ER-resident cargos bind to coatomer as monomers and to sites other than gamma-COP. The COPI coat therefore discriminates between p24 proteins and ER-resident proteins by differential binding involving distinct subunits.