Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries

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Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries

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Mariangela Stasi, Maria De Luca, Cecilia Bucci ∗ Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Lecce, Italy

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a r t i c l e

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a b s t r a c t

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Article history: Received 8 August 2014 Received in revised form 5 December 2014 Accepted 11 December 2014 Available online xxx

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Keywords: Protein–protein interaction Yeast two-hybrid assay High-throughput screening Rab proteins Membrane traffic

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1. Introduction

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Protein–protein interactions regulate biological processes and are fundamental for cell functions. Recently, efforts have been made to define interactomes, which are maps of protein–protein interactions that are useful for understanding biological pathways and networks and for investigating how perturbations of these networks lead to diseases. Therefore, interactomes are becoming fundamental for establishing the molecular basis of human diseases and contributing to the discovery of effective therapies. Interactomes are constructed based on experimental data present in the literature and computational predictions of interactions. Several biochemical, genetic and biotechnological techniques have been used in the past to identify protein–protein interactions. The yeast two-hybrid system has beyond doubt represented a revolution in the field, being a versatile tool and allowing the immediate identification of the interacting proteins and isolation of the cDNA coding for the interacting peptide after in vivo screening. Recently, variants of the yeast two-hybrid assay have been developed, including high-throughput systems that promote the rapidly growing field of proteomics. In this review we will focus on the role of this technique in the discovery of Rab interacting proteins, highlighting the importance of high-throughput two-hybrid screening as a tool to study the complexity of membrane traffic machineries. © 2014 Elsevier B.V. All rights reserved.

Cellular events are modulated by functional interactions between specific proteins. A single protein can interact with different, and often numerous, partners in the cell, thus regulating different processes. Although the human genome has been sequenced, many uncharacterized genes remain whose functions are unknown. Thus, identifying new interactions helps to characterize these genes, new protein functions and, possibly, new molecular pathways. Indeed, the role and function of a specific protein in a cell can often be inferred by identifying its molecular partners. Furthermore, the discovery of previously unknown interactions between known and characterized proteins may reveal new and possibly unexpected roles for these proteins. Several techniques are available to analyze protein–protein interactions. Biochemical approaches, such as co-immunoprecipitation and affinity chromatography followed by mass spectrometry, have been

∗ Corresponding author at: Department of Biological and Environmental Sciences and Technologies (DiSTeBA), University of Salento, Via Provinciale Lecce-Monteroni Q2 N. 165, 73100 Lecce, Italy. Tel.: +39 0832 298900; fax: +39 0832 298626. E-mail address: [email protected] (C. Bucci).

widely used with success, although the identification of interactors with these techniques is usually time consuming and laborious. Moreover, after the difficult process of protein purification and identification, the cDNA still must be obtained. The yeast twohybrid assay, developed in 1989 by Fields and Song, revolutionized the process of searching for interacting protein (Fields and Song, 1989). This technique allows rapid identification of several putative interacting proteins for a given protein of interest, enabling also the isolation of the cDNA associated with the interacting peptides (Chien et al., 1991). Similar versatility is present in the phage or virion display technique that was developed at the same time (McCafferty et al., 1990; Smith, 1985). In this case, cDNAs coding for peptides or for antibody fragments are inserted into viruses, and peptides or antibody fragments are expressed that are fused with viral coat proteins. In this manner, “displaying viruses” are generated and subsequently screened against proteins, peptides, DNA or other molecules, thus allowing for the detection of interactions (McCafferty et al., 1990; Smith, 1985). However, the great advantage of the two-hybrid system over the virion display and mass spectrometry techniques is that the screening is performed in vivo (Chien et al., 1991; Fields and Song, 1989). The two-hybrid system has beyond a doubt greatly contributed to interactome mapping (Parrish et al., 2006). Mass spectrometry and the

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Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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two-hybrid systems present similar limitations, such as the detection of spurious and non-physiological interactions, or the detection of only a limited number of interactions. However, these two techniques address different aspects of protein–protein interactions, and thus they are considered complementary. Indeed, while the two-hybrid system mainly identifies direct binary interactions, mass spectrometry can identify the components of a complex. Thus, combination of data coming from both approaches allows for a more complete and reliable map of interactions. Notably, the yeast two-hybrid method has been improved greatly in recent years, and several variants of the technique have been described that can search for membrane proteins, DNA-binding proteins and RNA binding proteins (Causier and Davies, 2002; Petschnigg et al., 2014; Reece-Hoyes and Marian Walhout, 2012). Furthermore, reverse two-hybrid systems that search for molecules that disrupt interactions have been developed and have been proven to be extremely useful for drug discovery (Fetchko et al., 2003; Vidal et al., 1996a). In this review we will focus on the yeast two-hybrid assay, describing the different variants that are now available, and will illustrate examples of its use. In particular, we will highlight the contribution of this technique to the identification of components of the membrane traffic machinery through the search of interactors of Rab GTPases. 2. Principle of the yeast two-hybrid assay The yeast two-hybrid assay is based on the modular nature of eukaryotic transcription factors. Indeed, transcription factors are structurally composed of domains: the DNA-binding domain (BD), which binds DNA sequences (called response elements) mediating the recruitment of the transcription factor on specific genome DNA sequences, the activation domain (AD), or trans-activating domain (TAD), which is responsible for recruitment of the transcription machinery, and an optional signal sensing domain (SSD), which is able to sense external signals and regulate the transcription complex (Latchman, 1997). The first two domains together are sufficient to initiate transcription, and it has been established that they do not need to be present within the same protein in order to function, although they have to be in close proximity (Ma and Ptashne, 1988). To develop the first two-hybrid systems, the properties of both domains were used, taking advantage of the fact that eukaryotic transcription factors are separable in BD and AD domains (Causier, 2004; Fields and Song, 1989). The system takes its name from the two hybrid proteins, one containing the BD domain and the other containing the AD domain, that are used to test the interaction. Indeed, if the two proteins interact, the AD and the BD components will be brought together, thus reconstituting the transcription factor and activating transcription of the reporter gene (Fig. 1A). Fields and Song developed the method using the BD and AD of the yeast GAL4 protein; however, domains from other transcription factors can also be used. Furthermore, the system can also function by combining BD and AD domains from different transcription factors (Chien et al., 1991; Fields and Song, 1989). For instance, the BD of the bacterial repressor protein LexA can be used with the AD of yeast GAL4, or with the AD of the Escherichia coli B42 transcription factor (Gyuris et al., 1993). 2.1. Monitoring interactions and selecting clones containing interacting proteins Normally, in the two-hybrid assay, the cDNA sequence encoding the protein of interest (called protein X) is sub-cloned into a plasmid vector that express the protein fused to the BD of a yeast transcription factor. This fusion protein is then used as a “bait” to

screen a cDNA library in order to identify protein X-interacting proteins called “preys”. The cDNA library is cloned into a plasmid that encodes the AD, leading to the expression of putative prey proteins (Fig. 1A). If protein X interacts with a prey, the BD and the AD domains reconstitute an active transcription factor, thus initiating transcription at reporter genes. The reporter genes used for this assay are genes that are required for the biosynthesis of amino acid and/or nucleotide precursors. The yeast strains used in the two-hybrid system are genetically engineered in a manner that the biosynthesis of certain nutrients is lacking. In particular, reporter genes have modified promoters that are recognized by the BD of the bait, and their transcription should begin only if the bait and the prey interact, thus reconstituting a transcription factor. The absence of interaction, and thus of transcription of these genes, prevents yeast growth as these metabolites are fundamental for growth and are not included in the culture medium. Instead, the physical interaction between bait and prey activates transcription and promotes the expression of the reporter gene that encode enzymes necessary for the synthesis of amino acid and/or nucleotide precursors. Common reporter genes include HIS3, LEU2, URA3, TRP1, LYS2 and ADE2. Therefore, the use of minimal media lacking these metabolites allows growth, and consequently selection, of the yeast clones where the transcription of the reporter(s) gene(s) was activated, possibly by bait–prey interaction. For example, expression of a HIS3 reporter is monitored by the growth of cell colonies on minimal medium lacking histidine (Causier, 2004; Coates and Hall, 2003). Often, in the two-hybrid system, multiple reporter genes under the control of promoters with similar response elements, but located in different part of the genome, are used in order to reduce the number of false positives. Another reporter gene used to further decrease false positives in the two-hybrid system is LacZ. This gene encodes the enzyme ␤galactosidase and it is not a nutritional selectable marker, but is rather a neutral screenable marker. Clones selected with nutritional markers are then tested for ␤-galactosidase activity, thus lowering the number of false positives (Bartel et al., 1993; Bartel and Fields, 1995). Furthermore, the use of this reporter gene allows quantification of the interaction if a colorimetric and spectrophotometric substrate for the ␤-galactosidase enzyme, which is able to measure enzyme activity, is used (Bartel et al., 1993; Bartel and Fields, 1995). 2.2. Limits of the system The two main limitations of the system are the possibility of having false negatives and false positives. Regarding false negatives, all proteins expressed in yeast have to fold properly, have to be correctly post-translationally modified, have to reach the nucleus and should not be toxic for yeast. Otherwise, not being able to interact with their partners, they will originate false negatives. For instance, the construction of a fusion protein leading to a different tertiary structure could mask or modify interacting domains, resulting in a false negative. However, the high number of false positive clones represents the main problem with the two-hybrid screening. Spurious activation of the reporter gene can occur for different reasons. For example, presence of preys able to bind DNA or able to activate reporter genes are the more frequent causes and can be counteracted by the use of more than one reporter gene and checked by swapping the BD and AD domains in the two proteins. Another frequent cause of false positives is due to interactions that cannot be confirmed in other experimental systems and, in particular, in the correct cell environment. The presence of these false positives can be due to the different environment where the interaction takes place (yeast versus mammalian, nucleus versus cytosol, for instance), or to the fact that proteins that normally are localized within different compartments and do not physiologically ever

Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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Fig. 1. General strategy for the one- two- and three-hybrid assay. The interaction between (A) protein X fused with the transcription BD and protein Y fused with the transcription AD in the two-hybrid assay, (B) DNA bait and protein Y fused with the transcription AD in the one-hybrid assay, and (C) RNA X linked to the transcription BD via a RNA binding domain and protein Y fused with the transcription AD in the three-hybrid assay which reconstitutes an active transcription factor that promotes recruitment of the transcription machinery and expression of the reporter gene.

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meet each other, in the two-hybrid system are brought together in the nucleus, where they may interact. For these reasons, once a putative interactor for a given protein has been identified, it is fundamental that the interaction be validated with other techniques (i.e., co-immunoprecipitation) and it must be demonstrated that both proteins are present in the same cellular compartment (Bartel et al., 1993; Causier, 2004; Causier and Davies, 2002; Coates and Hall, 2003). In spite of these limits, yeast two-hybrid assays have been extensively used to discover and map interactions, and several studies indicate that high-throughput two-hybrid techniques (see below) are suitable for mapping a significant portion of the human interactome and producing high quality data (Venkatesan et al., 2009; Yu et al., 2008).

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3. Two-hybrid variants

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After the first description of the two-hybrid system, several new variants based on the original concept have been developed and described. Each of them presents some advantages or different applications compared to the classical assay. The variants represent rapid and simple approaches to detect in vivo not only protein–protein but also DNA–protein and RNA–protein interactions and have been developed in organisms other than yeast in order to be more rapid and/or versatile (Table 1).

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3.1. The one-hybrid assay

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The one-hybrid screen represents an attractive approach for identifying genes encoding proteins that can bind to a DNA sequence of interest (Wang and Reed, 1993). In this method, multiple copies of the DNA sequence of interest (DNA bait) are cloned in a reporter plasmid upstream of a reporter gene (e.g., HIS3 or LacZ). This reporter plasmid is stably integrated into the yeast genome, generating a “DNA bait strain” that can be selected and isolated. The DNA bait strain is transformed with a library containing cDNAs that encode proteins (protein Y) fused with a strong transcriptional activation domain, usually the GAL4 activation domain (GAL4-AD). The reporter gene is expressed when the fusion protein interacts with the DNA bait, and thus yeast colonies can grow and be selected (Fig. 1B). The cDNAs are then isolated and sequenced to identify the encoded protein that binds the DNA bait (Carey et al., 2012; Deplancke et al., 2004). The one-hybrid assay is a straightforward, sensitive and rapid approach for identifying DNA-binding proteins under native in vivo conditions, considering that the expression of the protein in yeast allows acquisition of some of the post-translational modifications present in mammalian cells. However, this method has some

limitations, such as, for instance, the fact that an endogenous yeast activator protein could recognize the DNA bait, activating transcription and determining large numbers of false positives (Carey et al., 2012). Recently, high-throughput yeast one-hybrid assays have also been developed. These methods have been applied to delineate Caenorhabditis elegans gene regulatory networks (Deplancke et al., 2004, 2006; Vermeirssen et al., 2007) and to screen Arabidopsis gene promoters (Brady et al., 2011; Gaudinier et al., 2011). In addition, a gateway-compatible yeast one-hybrid assay provided a genecentered assay to identify novel human transcription factors that can bind to a DNA sequence of interest in order to map human regulatory interactions (Reece-Hoyes et al., 2011). 3.2. The three-hybrid assay The three-hybrid assay is a genetic method that is based on the yeast two-hybrid system, in which three chimeric molecules are expressed in yeast cells, and the interaction between these molecules induces the activation of a reporter gene. There are several types of three-hybrid assays. The RNA-based three-hybrid system allows rapid detection of RNA-binding proteins, while the ligand-based three-hybrid system searches for proteins that interact with different ligands (Becker et al., 2004; Cottier et al., 2011; Putz et al., 1996; SenGupta et al., 1996). Additionally, three-hybrid assays can be used to identify factors interacting with heteromeric proteins, and in fact have been used to identify physiological caspase-1 substrates and inhibitors (Van Criekinge et al., 1998). The RNA-based three-hybrid system is characterized by twohybrid protein molecules and a hybrid RNA molecule. The first hybrid protein consists of a DNA binding domain (for example, LexA-BD or Gal4-BD) that is present in multiple copies upstream from the reporter gene and is linked to an RNA binding domain (MS2-coat protein or Hiv-1 RevM10) that binds to a short stem-loop RNA sequence (protein X). The second hybrid protein is composed of an RNA binding protein (protein Y) and Gal4-AD. The two fusion proteins may be linked by a hybrid RNA molecule. The hybrid RNA is composed of the binding site for the first RNA binding protein (MS2 or RRE) and the RNA sequence of interest (RNA X). The interaction between RNA X and protein Y promotes functional transactivation of the reporter gene (Fig. 1C) (Bernstein et al., 2002; Jaeger et al., 2004; Putz et al., 1996; SenGupta et al., 1996). The method provides a powerful approach for examining RNA–protein interactions in vivo for a variety of purposes, including (i) testing of candidate RNA–protein pairs, (ii) characterization of known interactions through specific mutations, making it possible to identify single amino acid residues or domains and nucleotides important for the interaction (Lee and Linial, 2000; Martin et al., 2000;

Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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4 Table 1 Advantages and limits of the two-hybrid variants. Two hybrid variant (model organism)

Advantages

Limits

High-throughput screenings

“Classical” two-hybrid assay (yeast)

Straightforward, sensitive and rapid identification of protein–protein interactions. Straightforward, sensitive and rapid identification of DNA-binding proteins (Carey et al., 2012). In vivo detection of RNA–protein interactions and classification of RNA aptamers (König et al., 2007).

False positives and false negatives.

(Fukuda et al., 2011; Lindsay et al., 2013)

False positives (Carey et al., 2012).

(Gaudinier et al., 2011; Reece-Hoyes et al., 2011)

Lack of post-transcriptional modifications or cofactors; folding problems of the chimera; cut of RNA in the nucleus or translocation into other organelles (Jaeger et al., 2004). Membrane proteins are not identified (Jaeger et al., 2004); yeasts have the capacity to efficiently extrude some kind of drugs (Cottier et al., 2011). Difficulties in the creation of suitable mutants as the structure of several interactors is not known (Vidal and Legrain, 1999). Proteins may be toxic in bacteria; lack of post-translational modifications occurring in eucaryotic cells (Hu et al., 2000; Causier and Davies, 2002).

(König et al., 2007)

One-hybrid assay (yeast, bacteria and mammalian cells) Three-hybrid assay RNA based (yeast)

Three-hybrid assay ligand-based (yeast)

Reverse two-hybrid assay (yeast, bacteria and mammalian cells) Bacterial two-hybrid assay (bacteria)

Mammalian two-hybrid assay (mammalian cells)

Transcription-independent two-hybrid assays Membrane yeast two-hybrid assay (split-ubiquitin) (yeast) Mammalian-membrane two-hybrid assay (mammalian cells) Split-FP assay (yeast, bacteria and mammalian cells)

SRS assay (yeast and mammalian cells)

Split-TEV assay (mammalian cells)

Split-luciferase assay (yeast and mammalian cells)

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Identification of different kind of ligands as interactors of heteromeric proteins and candidate drug targets (Becker et al., 2004; Cottier et al., 2011; de Felipe et al., 2004). Further characterization of known interactions and screening of drugs (Fetchko et al., 2003; Leanna and Hannink, 1996; Vidal et al., 1996a,b; White, 1996). Faster, easier and with higher efficiency of transformation; reduction of false negatives; expression of proteins toxic for yeast; higher permeability to small molecules (Hu et al., 2000). Rapid and quantitative; protein interactions mimic better the in vivo situation (Lievens et al., 2012; Luo et al., 1997). Strong reduction of false positives and negatives. Identification of membrane protein interactions; reduced risk of alteration of the protein structures; detection of transient interactions (Stagljar et al., 1998). Extremely versatile (Petschnigg et al., 2014). No need of substrates or cofactors; sensitivity, flexibility and detection of weak interactions (Park et al., 2007; Blakeley et al., 2012). Detection of cytosolic interactions; detection of interactions in viruses that replicates in the cytoplasm (Moerdyk-Schauwecker et al., 2011). Sensitive, flexible, rapid and cost-effective detection of weak interactions (Lievens et al., 2009; Djannatian et al., 2011). Quantitative analysis of interactions (Stynen et al., 2012).

Ostrowski et al., 2002), and (iii) identification of new interactors of known RNA sequence and vice versa, using libraries of genomic and cDNA sequences in activation domain vectors and libraries of hybrid RNAs, each of which carries a different artificial or cellular sequence fused to coat protein binding sites (Bernstein et al., 2002; Sengupta et al., 1999). In addition, the combination of the SELEX technique with the three-hybrid system has been developed for the selection and classification of in vivo RNA aptamers (König et al., 2007). The three-hybrid assay is a powerful method for analyzing RNA–protein interaction in vivo. However, the range of RNA–protein interactions that can be assayed is limited. Indeed, the assay is performed using chimeric molecules, and folding of this chimera could prevent the interaction. In addition, the hybrid RNA could be cut in the nucleus or translocated into other organelles (Jaeger et al., 2004). Additionally, several RNAs require post-transcriptional modification or cofactors to bind to specific protein that are not present in yeast cells (Jaeger et al., 2004).

(Baker et al., 2003)

(Li et al., 2013)

(Paschos et al., 2011; Houot et al., 2012)

Less suitable for cDNA library screenings (Lievens et al., 2012; Luo et al., 1997).

(Fiebitz et al., 2008; Ravasi et al., 2004)

False positives (Fetchko and Stagljar, 2004).

(Lalonde et al., 2010; Kittanakom et al., 2014)

Slower and more complex.

(Petschnigg et al., 2014)

Photobleaching, phototoxicity, autofluorescence, low selectivity and difficult identification of subcellular localization (Stynen et al., 2012). False negatives and false positives (Moerdyk-Schauwecker et al., 2011).

(Pu et al., 2011)

Difficult to test interaction kinetics and dynamics; performance depends on cell line (Lievens et al., 2009; Djannatian et al., 2011). Difficult identification of subcellular localization (Fujikawa and Kato, 2007).

(Moerdyk-Schauwecker et al., 2011)

(Djannatian et al., 2011)

(Jester et al., 2010)

The ligand-based three-hybrid system has been successfully used to detect protein–small molecule interactions and is thus generally useful in the discovery of candidate drug targets having the necessary sensitivity (Becker et al., 2004; Cottier et al., 2011; de Felipe et al., 2004). 3.3. The reverse two-hybrid assay The main advantage of the two-hybrid system is the rapid identification of protein–protein interactions allowed by a genetic selection. However, subsequent validation and characterization of the interaction in order to establish its structural characteristics, functional meaning and regulation requires additional experimental work. A variant of the yeast two-hybrid system, referred to as the reverse two-hybrid assay, permits further characterization of the interactions by identifying the amino acids that are important for the interaction, as well other factors that disrupt specific protein–protein interactions (Fetchko et al., 2003; Leanna and

Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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Hannink, 1996; Vidal et al., 1996a,b; White, 1996). In this system, a reporter gene is used that leads to the production of a substance that is toxic for growing cells. An example is the URA3 gene, whose product is fundamental for uracil biosynthesis, but is also able to catalyze the formation of a toxic compound in the presence of 5-flouroorotic acid (5-FOA). Thus, when grown in complete media containing 5FOA, yeast cells with activated URA3 transcription will not be able to grow because of the production of the toxic compound. In contrast, the presence of a mutation disrupting the interaction will turn off URA3 leading to yeast growth (Vidal et al., 1996a,b). In this manner the systems screen for mutations, proteins or compounds able to disrupt a given interaction. Similarly, sensitivity/resistance to cycloheximide caused by the CYH2 gene can also be used (Leanna and Hannink, 1996). Therefore, reverse two-hybrid systems can be used to (i) identify proteins able to disrupt a specific interaction, (ii) screen randomly generated mutant proteins that are unable to bind to their molecular partner, (iii) screen cDNA libraries in order to identify proteins that regulate a specific protein–protein interaction, and (iv) screen for drugs that abolish specific protein–protein interactions (Fetchko et al., 2003; Leanna and Hannink, 1996; Vidal et al., 1996a,b; White, 1996). This variant of the two-hybrid system thus represents a very valuable tool for studying the regulation of protein–protein interactions.

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3.4. Bacterial and mammalian two-hybrid assay

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Although the original yeast two-hybrid and its variant one- and three-hybrid systems have been successful in detecting numerous interactions, modifications of the system in bacterial and mammalian cells have been developed to improve the detection of interactions (Caligiuri et al., 2006; Dove et al., 1997; Hu et al., 2000; Lievens et al., 2012; Luo et al., 1997). The advantages of using E. coli-based one- and two-hybrid systems include less time to conduct the screening, because bacteria grow faster, and higher efficiency of transformation of bacteria compared to yeast, thus allowing rapid analysis of larger libraries. Additionally, in the classical yeast two-hybrid system, various passages are conducted in E. coli (for instance construction and amplification of the library, transformation and amplification of the prey plasmids once isolated, etc.). Thus, using E. coli directly simplifies the procedures and requires less time by eliminating the need for shifting from yeast to E. coli, and then back to yeast. In addition, in bacterial two-hybrid assays, proteins do not have to enter a nucleus in order to interact, thus diminishing the number of false negatives by eliminating the requirement that one or both hybrid proteins enter the nucleus. Furthermore, the expression of some proteins could be toxic in yeast because of their role in the cell cycle or in proliferation, although toxicity in E. coli could occur for other reasons (Hu et al., 2000). Another variant of the yeast two-hybrid system is the mammalian two-hybrid system (Caligiuri et al., 2006; Lievens et al., 2012; Luo et al., 1997). The main advantage of the mammalian two-hybrid system is that mammalian protein interactions occur in a more physiological environment, thus better mimicking the in vivo situation. Indeed, folding and post-translational modifications, although similar, present differences between yeast and mammalian cells, and these differences could be responsible not only for false negatives but also for false positives. Thus, the mammalian two-hybrid system can also be used as an alternative method to rapidly confirm interactions that were identified with the yeast two-hybrid system in order to reduce the number of false positives (Lievens et al., 2012; Luo et al., 1997). In the mammalian two-hybrid system, a reporter gene that codes for chloramphenicol acetyltransferase (cat) is frequently used. The advantage of this reporter is that assays that measure CAT activity can be performed 48 h after transfection and are quantitative. Alternatively, in the yeast two-hybrid

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system, colonies grow in 3–5 days and then, in order to perform a quantitative ␤-galactosidase assay, they have to be cultured in liquid medium (Bartel et al., 1993; Bartel and Fields, 1995; Lievens et al., 2012; Luo et al., 1997).

4. Transcription-independent two-hybrid systems We have discussed the limitations of the two-hybrid system and, although it is not possible to define them exactly, the fact that the system is dependent on nuclear transcription is a major limitation. Indeed, false negatives can be attributed to proteins that are unable to enter the nucleus, while false positives can be observed for proteins that are transcriptional repressor or activators. Therefore, systems that are independent of transcription have been developed (Aronheim et al., 1997; Brachmann and Boeke, 1997; Johnsson and Varshavsky, 1994). The ubiquitin-based split protein sensor system is based on fusion proteins containing properly folded ubiquitin that are rapidly cleaved by ubiquitin proteases (Johnsson and Varshavsky, 1994). Furthermore, it has been demonstrated that N- and Cterminal portions of ubiquitin (Nub and Cub), even if expressed separately, are able to fold properly, thus reconstituting ubiquitin and leading to cleavage of fusion proteins by ubiquitin proteases (Johnsson and Varshavsky, 1994). To test these interactions, proteins are expressed fused with Nub or Cub, but with a mutated Nub that has a much lower affinity for Cub. Therefore, Nub and Cub can no longer reconstitute ubiquitin unless the proteins fused to Nub and Cub are capable of interacting and bringing the two ubiquitin domains in close proximity to each other (Johnsson and Varshavsky, 1994). Consequently, interactions between the two fusion proteins reconstitute ubiquitin, leading to cleavage by deubiquinating enzymes (DUBs). To detect the cleavage, which indicates an interaction, a reporter protein is attached to Cub and then released by cleavage (Johnsson and Varshavsky, 1994). The split-ubiquitin system has been adapted subsequently for the analysis of interactions between membrane proteins and their membrane or cytosolic partners and is referred to as the membrane yeast two-hybrid system (MYTH) (Snider et al., 2010; Stagljar et al., 1998). In particular, the system functions by using membrane proteins whose cytosolic domains are tagged with Cub and a mutated form of Nub (NubG) (Stagljar et al., 1998). Because ubiquitin proteases are present in the cytosol, it is important that Nub and Cub reconstitute ubiquitin in the cytosol. A membrane bait protein is tagged with Cub and a chimeric transcription factor, while a prey protein is tagged with NubG. If the two proteins interact, their two split-ubiquitin halves are brought into close proximity allowing reconstitution of ubiquitin. Cleavage will then occur and liberate the transcription factor that will activate transcription of the reporter gene(s) (Snider et al., 2010; Stagljar et al., 1998). A mammalian membrane two-hybrid assay (MaMTH), based on the membrane split-ubiquitin assay, has been developed recently (Petschnigg et al., 2014). This mammalian variant of the membrane split ubiquitin assay is very versatile because it can be conducted in virtually any cell line, and it is suitable for high-throughput screenings (Petschnigg et al., 2014). Furthermore, several other assays have been developed based on the “split” concept. In the split-GFP system, soluble selfassociating fragments of green fluorescent protein (GFP) can be used to tag and detect soluble and insoluble proteins and to measure their interactions (Cabantous et al., 2005). In the BiFC assay, non-fluorescent fragments of GFP are fused to interacting partners whose interaction allows reassembly and folding of GFP, allowing it to fluoresce (Barnard et al., 2008). In the split-TEV system, fragments of a tobacco etch virus (TEV) protease were engineered such that the protease regains activity only after

Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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assembly and folding of the two fragments and interaction of the fusion proteins (Wehr et al., 2006). This system is highly versatile, as it combines advantages of the split-enzyme and reporter genemediated assays (Wehr et al., 2006). In the split-luciferase system two domains of luciferase are fused to two interacting proteins. When proteins interact, luciferase fragments are brought close to each other and form a complemented luciferase, which produces a luminescent signal (Ozawa et al., 2001; Azad et al., 2014). Another transcription-independent assay that has been successfully used to identify interacting proteins is the Sos recruitment system (SRS). In this system, Sos activates the Ras GTPase. Indeed, the yeast guanyl nucleotide exchange factor (GEF) Cdc25 is recruited to the plasma membrane by activated signaling receptors to transduce the signal, activate Ras and promote growth. The Cdc25-2 mutant, which is unable to grow at 36 ◦ C, can be rescued by the mammalian GEF Sos. In the Sos recruitment system, Sos is fused to protein X, while a myristoylation signal is added to protein Y, which anchors the protein to the plasma membranes. As a result of an interaction between proteins X and Y, Sos will be recruited on the plasma membrane and will activate Ras, promoting yeast growth at 36 ◦ C (Aronheim et al., 1997).

5. High-throughput assays Since the first description of the two-hybrid system more than 20 years ago, the method has been used mainly to search for single molecule interactors. During this period of time, the contribution of the two-hybrid system to interactomics, and consequently to system biology, has been consistent, as hundreds of novel protein partners have been identified. In the past 10 years, the availability of genome sequences, and the increasing use of robotics and emerging technologies have contributed to the scale up of approaches leading to the development of high-throughput methods. In fact, the two-hybrid system can be easily automated for high-throughput studies, and two screening strategies have been described: the matrix or array approach, and the library approach (Brückner et al., 2009). In the matrix approach, interactions between sets of baits and sets of preys are detected. The advantage of this method is that baits are in defined positions, thus allowing for the rapid identification of prey, while the use of full length ORFs may not identify some interactions (false negative). In the library approach, baits are tested for interactions against libraries that also contain cDNA fragments, reducing the presence of false negatives, but increasing false positives (Brückner et al., 2009), Presently, the use of highthroughput two-hybrid screening is constantly increasing because, compared to other techniques, no specialized large equipment is required for screening, and the method is relatively inexpensive (Brückner et al., 2009; Fields, 2005; Lievens et al., 2012). Clearly, however, the problem of false positives in this type of screening, where tens or hundreds of baits are used, is important. To minimize this problem, large scale co-immunoprecipitations have been performed. Additionally, a comparison of results from independent approaches, such as phage display, can be of help. In addition, computational analysis can be applied to discard likely false positives (Brückner et al., 2009; Fields, 2005; Lievens et al., 2012). Basically, two-hybrid assays, including transcription dependent and independent variants, are suitable for high-throughput screenings (Table 1), although specific two-hybrid techniques have been developed for high-throughput screenings. For instance, a luminescence-based mammalian interactome (LUMIER) assay has been developed as an automated high-throughput technology to map protein–protein interaction networks systematically in mammalian cells (Barrios-Rodiles et al., 2005). Furthermore, the mammalian protein–protein interaction trap (MAPPIT) can detect modification-independent and phosphorylation-dependent

interactions in mammalian cells, thus placing interactions in their physiological context (Eyckerman et al., 2001). This system is based on an engineered receptor with JAK kinase that lacks the active STAT site. If a bait protein of interest is added to the cytoplasmic end of the receptor, and the prey is linked to an active STAT binding site, the interaction between bait and pray will lead to STAT activation which in turn will activate a reporter gene, such as luciferase or puromycin resistance (Eyckerman et al., 2001). In this system, the use of mammalian cells can reproduce the physiological conditions required for protein processing or activation, thus generating high quality interaction data (Eyckerman et al., 2001). The yeast two-hybrid assay and its transcription dependent and independent variants have been extensively used to construct interactomes in several species, and a number of studies have attempted to assess the quality of the interaction data obtained, validating for instance screening completeness, assay sensitivity, sampling sensitivity and precision (Venkatesan et al., 2009; Yu et al., 2008). The results of these multiple and complex analyses demonstrate that high-throughput yeast two-hybrid systems provide high-quality binary interaction data (Venkatesan et al., 2009; Yu et al., 2008). Indeed, orthogonal methods, such as LUMIER and MAPPIT, have been used to validate large-scale data (Venkatesan et al., 2009; Yu et al., 2008). Using these methods, comparison of two-hybrid data with data from the combination of affinity purification and mass spectrometry reveals that both sets of data are of high quality, although they essentially map networks with different topological and biological properties (Venkatesan et al., 2009; Yu et al., 2008).

6. A yeast two-hybrid assay to investigate membrane traffic machineries: identification and characterization of Rab protein interactors The yeast two-hybrid assay has contributed greatly to the identification of new factors involved in the regulation of membrane traffic. Indeed, since its development, an increasing number of proteins having an important role in vesicular membrane traffic have been identified using as baits known key factors of membrane traffic. The best example is represented by Rab proteins, small GTPases that regulate virtually all steps of vesicular traffic and define membrane identity (Pfeffer, 2013; Stenmark, 2009). In fact Rab proteins, cycling from their GTP-bound to their GDPbound forms and vice versa, are involved in the formation of a vesicle from a donor compartment, in the selection of the cargo of the vesicle, and in the movement of the vesicle on cytoskeletal elements in order to reach the target compartment (Bucci and Chiariello, 2006; Pfeffer, 2013; Stenmark, 2009). In addition, Rab proteins, by recruiting tethering and fusion factors, play a key role in tethering and fusion of vesicles and/or organelles with target compartments (Pfeffer, 2013; Stenmark, 2009). Furthermore, their presence on an organelle determines compartment identity and function, and may also regulate the order of events of membrane trafficking (Pfeffer, 2013). To accomplish these functions, each Rab protein has to interact with a number of molecular partners, and the discovery of new partners helps to define the underlying molecular mechanisms or lead to the identification of new roles and functions. An impressive number of Rab interactors have been identified in the past 20 years using the two-hybrid assay, but this system was also extremely helpful in further characterizing these interactions (Fig. 2). In fact, two-hybrid based systems were extensively used not only for the identification of new Rab interactors by screening cDNA libraries but also to confirm and further analyze previously demonstrated interactions. For instance, the two-hybrid system was used to determine if the Rab interactor or effector protein was

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6.1. The case of Rab4, Rab6 and Rab11 Examples of two-hybrid systems used to detect and characterize protein–protein interactions are reported for Rab4, Rab6 and Rab11 Q4 in Table 2. Screening of cDNA libraries not only led to the identification of several interactors for these proteins but also provided additional relevant information on the newly discovered interactions.

Fig. 2. Two-hybrid system and Rab proteins. The scheme indicates the different applications of the two-hybrid system used in the study of Rab proteins. The system can be used not only for the identification of new Rab interactors by screening cDNA libraries but also to confirm previously demonstrated interactions, to determine if binding is restricted to the GTP or GDP-bound form of a certain Rab using dominant negative and dominant positive mutants, to determine if the interaction is specific for a certain Rab, to quantify and compare the strength of different interactions, to test new interactions with binary assays and to define domains and amino acid residues required for interaction. In addition, high-throughput screenings can be performed using several Rabs as bait.

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able to bind to the GTP- or GDP-bound form of Rab using dominant negative and constitutively active mutants (see for instance Bielli et al., 2001; Cantalupo et al., 2001; de Graaf et al., 2004; Hales et al., 2001; Matsui et al., 2012). As with other G proteins, Rab proteins contain GTP-binding and hydrolysis domains that can be mutated to obtain dominant negative or dominant positive mutants. For instance, the mutation S/T to N in the GKS/T domain yields mutant proteins that bind GDP with much higher affinity than GTP, thus being mainly GDP-bound (inactive). The mutation Q to L in the DTAGQE domain, instead causes a significant reduction of the intrinsic GTPase activity, therefore, mutant proteins carrying this mutation will exist mainly in the GTP-bound (active) form. Similarly, a mutation of I to M in the domain responsible for GAP interaction reduces hydrolysis, generating a more active mutant protein. These mutant proteins have been very useful, not only for determining if the interacting protein was able to bind the GDP- or GTP-bound form of the Rab but also as baits for screening. In fact, in several cases the Rab dominant positive Q to L mutants have been used as bait for cDNA library screenings to identify proteins that bind active Rab proteins and to select for effector proteins. In addition, with the two-hybrid system it is possible to verify the specificity of the interaction. For this purpose the two-hybrid assay was used to test the interactions between the identified prey and different Rab proteins to establish if the interaction was specific for a single Rab or for a subset of Rab proteins (see for instance Dong et al., 2004; Mori et al., 2013; Yuasa et al., 2008). In other cases, it has been useful to compare the strength of different interactions by measuring beta-galactosidase activity, although this method is not considered to be completely reliable. For instance, in order to identify the domains or the amino acid residues involved in the interaction, deletion or site-directed mutants of the interactor and/or of the Rab protein have been created and tested in the two-hybrid assay using a quantitative ␤-galactosidase assay (see for instance Cantalupo et al., 2001; de Graaf et al., 2004; Horgan et al., 2013; Lindsay et al., 2013; Weide et al., 2001).

6.1.1. Rab4 Using two-hybrid screening of cDNA libraries, Rab4 has been found to interact with (i) the central region of cytoplasmic dynein light intermediate chain-1 (Dyn LIC-1), a retrograde motor protein, indicating that Rab4 recruits this motor on vesicles that must move in a retrograde manner on microtubules (Bielli et al., 2001); (ii) a FYVE-finger-containing protein, Rabip4, that is important for endocytic recycling, thus confirming the role of Rab4 in regulating recycling and providing information on the molecular mechanism of action (Cormont et al., 2001); (iii) a plasma membrane glycoprotein, P-gp, suggested to be a primary contributor to multidrug resistance in tumors; Rab4 controls trafficking of P-gp, suggesting that it may control P-gp temporal and spatial distribution (Ferrándiz-Huertas et al., 2011); (iv) RCP, a member of the Rab11FIP family, involved in endocytic recycling (Lindsay et al., 2002); and (v) Rabaptin-5, a protein that binds Rab5 in the C-terminal region and binds Rab4 at the N-terminus, thus coordinating endocytic trafficking and recycling (Vitale et al., 1998). The two-hybrid system was also used to demonstrate that the interaction between Dyn LIC-1, Rabip4 and RCP preferentially involved the GTP-bound form of Rab4 (Bielli et al., 2001; Cormont et al., 2001; Lindsay et al., 2002) and to identify the Rab4 binding domain of Rabip4 (Cormont et al., 2001). Furthermore, a highthroughput two-hybrid approach was used to screen a collection of chlamydial inclusion membrane proteins (Incs) against Rab4, and detected an interaction between the chlamydial inc CT229 and Rab4 (Rzomp et al., 2006). Further experiments demonstrated that CT229 recruits, on the inclusion membranes containing the bacteria, Rab4, suggesting a role for Rab4 in the regulation of chlamydial inclusion trafficking and fusions (Rzomp et al., 2006). 6.1.2. Rab6 Two-hybrid screenings of cDNA libraries have also contributed to the identification of several Rab6 interacting proteins such as BICD1 (Matanis et al., 2002), Rab6IP2A and Rab6IP2B (Monier et al., 2002), DYNLRB1 (Wanschers et al., 2008), Mint1 826 (Thyrock et al., 2013), Mint 3 (Teber et al., 2005), NSF (Han et al., 2000) and Giantin (Rosing et al., 2007). Thus, Rab6 interacts directly with the dynein light chain protein DNLRB1 but also recruits Bicaudal D1 (BICD1) that, in turn, recruits the dynein–dynactin complex controlling retrograde Golgi to ER transport (Matanis et al., 2002; Wanschers et al., 2008). It was also shown that Rab6 regulates trafficking and processing of amyloid precursor protein (APP), interacting with the adaptor proteins mint1 826 and mint3 that are implicated in the amyloidogenic pathway (Teber et al., 2005; Thyrock et al., 2013). The direct interaction of Rab6 with the N-ethylmaleimidesensitive fusion protein (NSF) suggests that Rab6 controls vesicle fusion through NSF, while the interaction with giantin, a Golgi protein important for Golgi vesicle fusion, for Golgi disassembly and reassembly in mitosis, and for Golgi structure, indicates a role for Rab6 in the structural maintenance of this compartment and in the regulation of its functions (Han et al., 2000; Rosing et al., 2007). The two-hybrid system also established that the interaction between mint3 and Rab6 was preferentially with the GTP-bound form of Rab6 (Teber et al., 2005), and demonstrated that the NSF binding site is at the C-terminus of Rab6 (Han et al., 2000). A highthroughput two-hybrid screening using all Rabs discovered the

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Q8 Examples of data obtained by two-hybrid systems on Rab4, Rab6 and Rab11 proteins. Rab protein

Rab4

Rab6

Rab11

Applications of the two-hybrid system in the study of Rab proteins Screening of cDNA libraries

Nucleotide binding

High-throughput

Binding residues/domains

Dyn LIC-1 (Bielli et al., 2001) RabIP4 (Cormont et al., 2001) P-gp (Ferrándiz-Huertas et al., 2011) RCP (Lindsay et al., 2002)

GTP bound/Dyn LIC-1 (Bielli et al., 2001)

Screening of a collection of chlamydial Incs to test their ability to interact with Rab4 (Rzomp et al., 2006)

Rabip4 507–517 (Cormont et al., 2001)

Screening of all human Rabs to test the interaction with MyosinVa. Interaction Rab6/MyosinVa (Lindsay et al., 2013)

C-terminus of Rab6 is responsible for the binding to NSF (Han et al., 2000)

GTP bound/Rabip4 (Cormont et al., 2001)

GTP-bound/RCP (Lindsay et al., 2002)

BICD1 (Matanis et al., 2002) Rab6IP2A (Monier et al., 2002) Rab6IP2B (Monier et al., 2002) DYNLRB1 (Wanschers et al., 2008) Mint1 826 (Thyrock et al., 2013) Mint3 (Teber et al., 2005) NSF (Han et al., 2000) Giantin (Rosing et al., 2007)

GTP-bound/mint3 (Teber et al., 2005)

Gbetagamma (García-Regalado et al., 2008) MyosinVb (Lapierre et al., 2001) PI4K␤ (de Graaf et al., 2004) Rab11-FIP1 (Hales et al., 2001) hIP (Wikström et al., 2008) RCP (Lindsay et al., 2002)

GTP-bound/PI4K␤ (de Graaf et al., 2004)

Screening of 60 different Rabs for the interaction with the RUN domains of 19 different RUN domain-containing proteins. Interaction DENND5A/Rab6A (Fukuda et al., 2011)

GTP-bound/Rab11-FIP1 (Hales et al., 2001)

TBC domain of Evi5 was used to test interaction against a panel of GTPase-deficient Rab GTPases. Interaction Evi5/Rab11 (Westlake et al., 2007)

hIP 299–320 (Wikström et al., 2008)

Rab11-FIP1 615–632 (Hales et al., 2001)

The two-hybrid system was used to screen cDNA libraries in order to find new interacting proteins, to perform high-throughput assays, to test interaction with the GTPor GDP-bound form of the Rab and to identify domains or amino acid residues involved in the interaction. Abbreviation used: Dyn LIC-1, dynein light intermediate chain1; Rabip4, Rab4-interacting protein; P-gp, P-glycoprotein; RCP, Rab coupling protein; Incs, chlamydial inclusion membrane proteins; BICD1, Bicaudal-D; Rab6IP2A, Rab6 interacting protein 2A; Rab6IP2B, Rab6 Interacting Protein 2B; DYNLRB1, dynein light chain roadblock-type 1; Mint1 826, Amyloid beta A4 precursor protein-binding family A member 1; Mint3, amyloid beta A4 precursor protein-binding family A member 3; NSF, N-ethylmaleimide-sensitive fusion protein; DENND5A, DENN domain-containing protein 5A; Evi5, Ecotropic viral integration site 5 protein homolog; PI4K␤, phosphatidylinositol 4-kinasebeta; Rab11-FIP1, Rab11-family interacting protein1; hIP, human prostacyclin receptor. 660 661 662 663 664 665

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interaction of Rab6 proteins (Rab6 and isoforms) with Myosin Va (Lindsay et al., 2013). Another high-throughput screening using approximately 60 Rabs and 19 different RUN domains (domains involved in the regulation of Rab GTPases) revealed several interactions, one of which being between Rab6 and DENND5A/B (Fukuda et al., 2011). 6.1.3. Rab11 Two-hybrid screenings of cDNA libraries identified Gbetagamma, Myosin Vb, PI4K␤, Rab11-FIP1, hIP and RCP as Rab11 interactors (de Graaf et al., 2004; García-Regalado et al., 2008; Hales et al., 2001; Lapierre et al., 2001; Lindsay et al., 2002; Wikström et al., 2008; Reid et al., 2010). The discovery of the interaction between Rab11 and Gbetagamma revealed that Gbetagamma, via its interaction with Rab11, causes endosomal activation of PI3K/AKT signaling, and thus Rab11, regulating Gbetagamma trafficking, is able to modulate this signal transduction pathway (García-Regalado et al., 2008). The interaction with myosin Vb revealed that myosin Vb, together with Rab11, is associated with the recycling pathway, thus regulating this transport step (Lapierre et al., 2001). Interestingly, the interaction between phosphatidylinositol 4-kinase beta PI4K␤ revealed that this protein acts as a docking protein for Rab11 in the Golgi because inhibition of Rab11 binding to PI4K␤ abolishes Golgi localization of Rab11 (de Graaf et al., 2004). Using the two-hybrid assay, a family of Rab11-interacting proteins, Rab11-FIP, has been discovered, and its members, such as Rab11-FIP1, Rab11-FIP2 and Rab11-FIP3, have been characterized (Hales et al., 2001).

Two-hybrid assays also determined that PI4K␤ and Rab11-FIP1 preferentially bind the GTP-bound form of Rab11, while Rab11FIP2 also interacted with the GDP-bound form (de Graaf et al., 2004; Hales et al., 2001). High-throughput screening using a panel of Rab GTPases against the Evi5 oncogene revealed its interaction with Rab11, leading to the notion that Rab11 and Evi5 coordinate not only vesicular intracellular trafficking but also the cell cycle (Westlake et al., 2007). Furthermore, the use of two-hybrid assays identified the Rab11 binding domains of hIP and Rab11-FIP1 (Hales et al., 2001; Wikström et al., 2008; Reid et al., 2010). 6.2. An example of Rab high-throughput two-hybrid screening High-throughput two-hybrid screenings are extremely powerful tools for searching and characterizing interactions, and several Rabs (e.g., all human Rabs) might be used as baits against one or more preys or a cDNA library. An interesting recent example regards the screening of all human Rabs to study interactions with myosin Va (Lindsay et al., 2013). Indeed, a yeast two-hybrid screen was performed using constitutively active and dominant negative forms of each human Rab as baits, and the tail region of myosin Va as prey (Lindsay et al., 2013). This assay identified 10 new Rab proteins that interact with myosin Va, namely, Rab 3B, 3C, 3D, 6A, 6A , 6B, 11B, 14, 25, 39B, in addition to the previously reported interactions with Rab3A, Rab10 and Rab11A (Lindsay et al., 2013; Roland et al., 2009; Wollert et al., 2011). In this study, it was also established that Myosin Va interacts preferentially with the GTP-bound form of these Rab proteins and is able to interact only with the

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Fig. 3. An example of high-throughput screening using Rab proteins as bait. The two-hybrid system is a useful tool for detecting new interactions by screening panels of proteins using a single prey. In this example, all human Rabs were screened against myosinVa. The screening led to the identification of ten new Rab proteins (Rab3B, 3C, 3D, 6A, 6A , 6B, 11B, 14, 25, and 39B) interacting with myosin Va and confirmed the interaction with the previously reported interactors Rab3A and Rab11A. The new Rab proteins were part of three different Rab subfamilies (Rab6, Rab14, Rab39B).

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constitutively active mutants and that the Rab proteins interacting with Myosin Va belong to at least five distinct subfamilies: Rab3 (Rabs 3A, 3B, 3C and 3D), Rab6 (Rabs 6A, 6A and 6B), Rab11 (Rab11A, Rab11B and Rab25), Rab14 and Rab39B, three of which were newly discovered (Rab6, Rab14, Rab39B) (Fig. 3) (Lindsay et al., 2013; Roland et al., 2009; Wollert et al., 2011). Furthermore, the study demonstrated the presence of three distinct Rab binding domains in Myosin Va (Lindsay et al., 2013). These data indicate that two-hybrid high-throughput screenings using Rab proteins as baits can be extremely informative, as they define subfamilies of Rabs interacting with a given protein. Additional two-hybrid tests can then be used to determine features of the interaction as, for instance, interacting domains.

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The two-hybrid system was extensively used to identify and characterize Rab protein interactors. To provide a clear understanding of how powerful this technique is for the study of Rab proteins in general, and for a given Rab in particular, we focused on the findings obtained with one Rab protein, Rab7. Numerous Rab7 interacting proteins have been identified and most of them have actually been found using the two-hybrid assay (Fig. 4). Rab7 regulates late endocytic trafficking, being responsible for traffic to late endosomes and lysosomes, to phagolysosomes and to autolysosomes (Bucci et al., 2000; Harrison et al., 2003; Jager et al., 2004; Vitelli et al., 1997). The discovery of Rab7 interacting proteins has been important in defining the molecular mechanism of action of this GTPase, and has also shed light on the multiple functions of this GTPase. The first Rab7 interactor identified by a two-hybrid screening was the prenylated Rab acceptor (PRA1), a member of the Yip family that binds to several Rab proteins and catalyzes the dissociation of endosomal Rab from GDI (GDP Dissociation Inhibitor) (Bucci et al., 1999; Sivars et al., 2003). In 2001 the Rab-interacting lysosomal protein RILP was identified as a Rab7 effector responsible for recruiting, on Rab7-positive organelles, dynein–dynactin complexes (Cantalupo et al., 2001; Jordens et al., 2001). Functional studies established that Rab7 and

RILP together regulate transport to lysosomes and phagolysosomes (Cantalupo et al., 2001; Harrison et al., 2003; Jordens et al., 2001). Subsequently RILP, again using the two-hybrid assay, has been shown to interact also with Rab34 and Rab36 (Matsui et al., 2012; Wang and Hong, 2002). The oxysterol binding protein related protein 1L (ORP1L) has been found to interact with Rab7 and to form a complex with RILP that, simultaneously, also binds the p150(Glued) subunit of the dynein motor and the tethering HOPS complex (Johansson et al., 2005; Johansson and Olkkonen, 2005; Rocha et al., 2009; van der Kant et al., 2013). In this complex, ORP1L senses cholesterol and controls interactions regulating retrograde transport on microtubules and fusion (Johansson et al., 2005; Johansson and Olkkonen, 2005; Rocha et al., 2009; van der Kant et al., 2013). Rubicon and PLEKHM1 interact directly with Rab7 via their RH domains (Tabata et al., 2010). Rubicon regulates endocytosis, and its interaction with Rab7 promotes loading of GTP-bound Rab7 on endosomes, thus stimulating endosomal maturation (Sun et al., 2010; Tabata et al., 2010). XAPC7/PSMA7 is a proteasome subunit that is recruited on the membrane of late endosomes by direct interaction with Rab7 (Dong et al., 2004). XAPC7 overexpression caused decreased late endosomal transport that was partially rescued by Rab7 overexpression (Dong et al., 2004). The discovery of this interaction revealed a close link between endocytic structures and cytosolic degradative machineries (Dong et al., 2004). Rabring7 is recruited by GTP-bound Rab7 on late endosomes and lysosomes. Overexpression of Rabring7 affects EGFR degradation and causes perinuclear accumulation of late endosomes and lysosomes, indicating its important role in late endocytic traffic (Mizuno et al., 2003). The interaction of Rab7 with Rac1, a small GTPase regulating actin cytoskeleton, suggests that Rab7 acting on Rac1 could regulate movement of endosomes on actin filaments and/or actin organization (Sun et al., 2005). A direct interaction between GTP-bound Rab7 and neuronal ceroid lipofuscinosis protein CLN3 has been reported (Uusi-Rauva et al., 2012). CLN3 also interacts with RILP (Uusi-Rauva et al., 2012). Expression of a CLN3 disease-causing mutant affects the

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Fig. 4. Scheme of Rab7 interactors. The figure shows the known Rab7 interacting proteins identified using the two-hybrid system (rectangles). In several cases these interactions were discovered by screening cDNA libraries and then confirmed using binary two-hybrid assays (yellow) or other methods (cyan). In other cases, interactions were discovered using binary two-hybrid assays (orange rectangles), yeast three-hybrid system (green), bacterial (red) or mammalian (pink) two-hybrid system. Few Q7 interactions (circles) were established using other techniques including co-immunoprecipitation, affinity purification or crystallography. RILP, Rab-interacting lysosomal protein; PSMA7, proteasome subunit alpha type-7; ORP1L, oxysterol binding protein related protein 1L; CLN3, ceroid lipofuscinosis neuronal 3; PCBP1, Poly(RC) binding protein 1; PRA1, prenylated Rab acceptor protein 1; PLEKHM1, pleckstrin homology domain-containing family M member 1; Mon1-CCZ1, Vacuolar fusion protein MON1 (Monensin sensitivity protein 1)-vacuolar fusion protein CCZ1 (Calcium Caffeine Zinc Sensitivity 1); RAC1, Ras-related C3 botulinum toxin substrate 1; VPS39, vacuolar protein sorting 39; VPS34, vacuolar protein sorting 34; FYCO, FYVE and coiled-coil domain-containing protein 1; CHM, choroideremia protein, also known as REP-1, Rab escort protein 1; NTRK1/TRKA, neurotrophic tyrosine kinase receptor type 1; CIN85-Dyn2, Cbl-interacting protein of 85 kDa – Dynamin-2. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

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late endosomal compartment causing perinuclear clustering of late endosomes and relocalization of Rab7 (Uusi-Rauva et al., 2012). Recently, two intermediate filament proteins (vimentin and peripherin) have been identified as Rab7 interacting proteins, thereby uncovering a role for Rab7 in the regulation of the intermediate filament cytoskeleton (Cogli et al., 2013a,b). Furthermore, because peripherin is predominantly expressed in peripheral neurons, this interaction could explain how mutations in Rab7, a ubiquitous protein, causes Charcot-Marie-Tooth type 2B disease, a peripheral neuropathy (Bucci and De Luca, 2012; Cogli et al., 2013b). In yeast Ypt7, the mammalian homolog of Rab7, has been observed to interact with VPS39, a protein involved in vacuolar sorting (Wurmser et al., 2000). VPS39 appears to interact with the GDP-bound and nucleotide-free form of Ypt7, thus stimulating nucleotide exchange, although this finding has been questioned (Nordmann et al., 2010; Wurmser et al., 2000). However, VPS39 surely acts as a Ypt7 effector, and tethers transport to vacuoles (Price et al., 2000; Wurmser et al., 2000). The endosomal protein CCZ1 interacts with Rab7 and with its partner Mon1. The Mon1-CCZ1 complex functions as GEF for Ypt7 triggering of endosomal maturation by activating Ypt7 on late endosomes (Nordmann et al., 2010). Interaction of Rab7 with PCBP1, a heterogeneous nuclear ribonucleoprotein binding RNA, has also been reported, although the physiological significance of this interaction is currently unknown (Huo et al., 2009). A number of other Rab7 interacting proteins, such as C9ORF72, CHM/REP, CIN85Dyn2, FYCO1, TrkA and VPS34/p150, have been discovered using co-immunoprecipitation, affinity purification or other methods (Farg et al., 2014; Pankiv et al., 2010; Rak et al., 2004; Saxena et al., 2005; Schroeder et al., 2010; Stein et al., 2003). We believe that employment of the two-hybrid assay could be fundamental for characterizing further the interactions between these proteins. Indeed, it could be used to determine domains of interactions, to demonstrate if the interacting protein binds to the GTP- or GDP-form of the Rab, and to establish if the interaction is specific for Rab7, or involves other Rab proteins of the same subfamily or of different subfamilies.

Notably, for a number of Rab7 interacting proteins, two-hybrid screenings led to the identification of new partners, thus enriching the interaction network. As an example, RILP has been found to interact with VPS22 regulating the formation of multivesicular bodies (Progida et al., 2006, 2007). Also RILP has been found to interact with the V1G1 subunit of the vacuolar ATPase (De Luca et al., 2014). In fact, Rab7 recruits membrane RILP that, in turn, recruits V1G1, and the Rab7/RILP ratio appears to affect V1G1 abundance and regulates localization and function of the vacuolar ATPase (De Luca et al., 2014). In conclusion, the discovery of Rab7 interacting proteins, and the identification of new partners for these interacting proteins, revealed a complex network of interactions involving Rab7 that is useful in defining the multiple roles of this GTPase in the late endocytic pathway.

7. Conclusions The yeast two-hybrid method was first developed in 1989 and was immediately recognized as an extremely powerful technique for discovering and investigating protein–protein interactions. Numerous modifications of the systems have been subsequently developed, allowing for the discovery and analysis of different types of interactions and proving extremely useful for drug discovery. The two-hybrid technology has provided fundamental contributions for interactome maps, and although 25 years have passed since its first description, it is increasingly used in all fields of biology, and its applications are expected to dramatically increase our knowledge of network interactions present in the cells, and for regulating different cell processes. In particular, the two-hybrid system contributed to the identification of numerous components of membrane traffic machineries and has defined Rab protein interactomes. High-throughput screening surely represents the future of this technique and will probably contribute in the coming years to revealing new interaction networks and will contribute to our understanding of molecular mechanisms underlying vesicular trafficking and other cellular processes.

Please cite this article in press as: Stasi, M., et al., Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries. J. Biotechnol. (2014), http://dx.doi.org/10.1016/j.jbiotec.2014.12.007

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