A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided.

Oligomerization into multimeric complexes is a prerequisite for the chaperone function of almost all α‐crystallin type heat shock proteins (α‐Hsp), but the molecular details of complex assembly are poorly understood. The α‐Hsp proteins from Bradyrhizobium japonicum are suitable bacterial models for structure‐function studies of these ubiquitous stress proteins. They fall into two distinct classes, A and B, display chaperone activity in vitro and form oligomers of ≈ 24 subunits. We constructed 19 derivatives containing truncations or point mutations within the N‐ and C‐terminal regions and analyzed them by gel filtration, citrate synthase assay and coaffinity purification. Truncation of more than the initial few amino acids of the N‐terminal region led to the formation of distinct dimeric to octameric structures devoid of chaperone activity. In the C‐terminal extension, integrity of an isoleucine‐X‐isoleucine (I‐X‐I) motif was imperative for α‐Hsp functionality. This I‐X‐I motif is one of the characteristic consensus motifs of the α‐Hsp family, and here we provide experimental evidence of its structural and functional importance. α‐Hsp proteins lacking the C‐terminal extension were inactive, but still able to form dimers. Here, we demonstrate that the central α‐crystallin domain alone is not sufficient for dimerization. Additional residues at the end of the N‐terminal region were required for the assembly of two subunits.

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AMP-activated protein kinase (AMPK) is a cellular and whole body energy sensor with manifold functions in regulating energy homeostasis, cell morphology and proliferation in health and disease. Here we apply multiple, complementary in vitro and in vivo interaction assays to identify several isoforms of glutathione S-transferase (GST) as direct AMPK binding partners: Pi-family member rat GSTP1 and Mu-family members rat GSTM1, as well as Schistosoma japonicum GST. GST/AMPK interaction is direct and involves the N-terminal domain of the AMPK β-subunit. Complex formation of the mammalian GSTP1 and -M1 with AMPK leads to their enzymatic activation and in turn facilitates glutathionylation and activation of AMPK in vitro. GST-facilitated S-glutathionylation of AMPK may be involved in rapid, full activation of the kinase under mildly oxidative physiological conditions.

The classical yeast two-hybrid system and its modifications have been successfully used over the past decade to investigate interactions between most classes of proteins expressed in a given cell or tissue. However, some proteins (e.g., integral membrane proteins or nuclear proteins) are relatively difficult to investigate by standard yeast two-hybrid methods either because they are retained at cellular membranes or they activate the system in the absence of a true protein interaction. The membrane-based yeast two-hybrid (MbY2H) system presented in this unit overcomes some of these limitations. It is based on the split-ubiquitin protein complementation assay and detects protein interactions directly at the membrane, thereby allowing the use of full-length integral membrane proteins and membrane-associated proteins as baits to hunt for novel interaction partners. A simple modification also allows the use of proteins that are self-activating in a classical yeast two-hybrid system (e.g., acidic proteins and many transcription factors). Like the yeast two-hybrid system, the MbY2H system can also be used for interaction discovery by screening complex cDNA libraries for novel interaction partners.