Identification and characterization of protein subcomplexes in yeast

Protein complexes are major components of cellular organization. Based on large-scale protein complex data, we present the first statistical procedure to find insightful substructures in protein complexes: we identify protein subcomplexes (SCs), i.e., multiprotein assemblies residing in different protein complexes. Four protein complex datasets with different origins and variable reliability are separately analyzed. Our method identifies well-characterized protein assemblies with known functions, thereby confirming the utility of the procedure. In addition, we also identify hitherto unknown functional entities consisting of either functionally unknown proteins or proteins with different functional annotation. We show that SCs represent more reliable protein assemblies than the original complexes. Finally, we demonstrate unique properties of subcomplex proteins that underline the distinct roles of SCs: (i) SCs are functionally and spatially more homogeneous than complete protein complexes (this fact is utilized to predict functional roles and subcellular localizations for so far unannotated proteins); (ii) the abundance of subcomplex proteins is less variable than the abundance of other proteins; (iii) SCs are enriched with essential and synthetic lethal proteins; and (iv) mutations in SC-proteins have higher fitness effects than mutations in other proteins.     

Identification of extracellular signal-regulated kinase 3 as a new interaction partner of cyclin D3

Cyclin D3, like cyclin D1 and D2 isoforms, is a crucial component of the core cell cycle machinery in mammalian cells. It also exhibits its unique properties in many other physiological processes. In the present study, using yeast two-hybrid screening, we identified ERK3, an atypical mitogen-activated protein kinase (MAPK), as a cyclin D3 binding partner. GST pull-down assays showed that cyclin D3 interacts directly and specifically with ERK3 in vitro. The binding of cyclin D3 and ERK3 was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis. Moreover, carboxy-terminal extension of ERK3 was responsible for its association with intact cyclin D3. These findings further expand distinct roles of cyclin D3 and suggest the potential activity of ERK3 in cell proliferation.     

Identification of a cDNA clone encoding DIP1-binding protein in Drosophila melanogaster

The Drosophila melanogaster L27a gene encodes a ribosomal protein which is a member of the L15 family of ribosomal proteins. D.m. L27a is closely related to the mammalian protein that has been found differentially expressed in lung cancer tissues and therefore could be involved in the control of cell proliferation such as the ribosomal protein S6. Our work elucidates the role of DIP1 which is a novel protein that we found in Drosophila. We performed a two-hybrid system assay and identified the L27a protein as an interactor of DIP1. The interaction was then validated by in vitro binding assays. DIP1, similar to other nuclear proteins in eukaryotes, is localized to the nuclear periphery and chromatin domain in all nuclei, but disappears at the metaphase. It is possible that in D.m. L27a protein, via interaction with DIP1, could be involved in protein synthesis as well as in cell cycle regulation.     

Identification of a hypothetical membrane protein interactor of ribosomal phosphoprotein P0

The ribosomal phosphoprotein P0 of the human malarial parasitePlasmodium falciparum (PfP0) has been identified as a protective surface protein. InDrosophila, P0 protein functions in the nucleus. The ribosomal function of P0 is mediated at the stalk of the large ribosomal subunit at the GTPase centre, where the elongation factor eEF2 binds. The multiple roles of the P0 protein presumably occur through interactions with other proteins. To identify such interacting protein domains, a yeast two-hybrid screen was carried out. Out of a set of sixty clones isolated, twelve clones that interacted strongly with both PfP0 and theSaccharomyces cerevisiae P0 (ScP0) protein were analysed. These belonged to three broad classes: namely (i) ribosomal proteins; (ii) proteins involved in nucleotide binding; and (iii) hypothetical integral membrane proteins. One of the strongest interactors (clone 67B) mapped to the gene YFL034W which codes for a hypothetical integral membrane protein, and is conserved amongst several eukaryotic organisms. The insert of clone 67B was expressed as a recombinant protein, and immunoprecipitaion (IP) reaction with anti-P0 antibodies pulled down this protein along with PfP0 as well as ScP0 protein. Using deletion constructions, the domain of ScP0, which interacted with clone 67B, was mapped to 60–148 amino acids. It is envisaged that the surface localization of P0 protein may be mediated through interactions with putative YFL034W-like proteins inP. falciparum     

Genetic characterization of Drosophila Mi-2 ATPase

Zinc-fingers and homeoboxes 1 (ZHX1) is a protein which interacts with the activation domain of the A subunit of nuclear factor-Y. To analyze the physiological role(s) of ZHX1, we searched ZHX1-interacting protein(s) using a yeast two-hybrid system. The rat counterpart of ZHX1 cDNAs was cloned from an ovarian granulosa cell complementary DNA (cDNA) library, indicating that ZHX1 is able to form a homodimer. An analysis of the nucleotide sequence and its deduced amino acid sequence show that rat ZHX1 consists of 873 amino acid residues. Northern blot analysis shows that ZHX1 messenger RNA is expressed ubiquitously and that the level in the ovary are not regulated by gonadotropins. Furthermore, transfection experiments with green fluorescence protein (GFP) expression vectors into human embryonic kidney HEK293 cells reveal that full-length ZHX1 fused to the GFP is localized in the nuclei. Thus, we report on the molecular cloning, expression and characterization of full-length rat ZHX1 cDNA.     

Identification and characterization of NIF3L1 BP1, a novel cytoplasmic interaction partner of the NIF3L1 protein

The NIF3L1 protein is strongly conserved during evolution from bacteria to mammals and recently its function in neuronal differentiation has been demonstrated. In the present study we identified novel binding partners of human NIF3L1 by screening a HeLa cDNA-library using the yeast two-hybrid system. We could show that the NIF3L1 protein is interacting with itself and with the NIF3L1 binding protein 1 (NIF3L1 BP1), a novel protein of 23.67kDa bearing a putative leucine zipper domain. Furthermore, both interactions were confirmed using the mammalian two-hybrid system. Deletion analyses clearly demonstrated that a C-terminal region of 100 amino acids of the NIF3L1 BP1 is sufficient for the interaction with NIF3L1. The NIF3L1 BP1 is ubiquitously expressed and cotransfection experiments revealed that NIF3L1 and NIF3L1 BP1 interact in the cytoplasm of human LNCaP cells. This study provides novel insights into the cellular function of the NIF3L1 protein.     

Application of Mu in vitro transposition for high-precision mapping of protein–protein interfaces on a yeast two-hybrid platform

High-precision mapping of regions involved in protein–protein interfaces of interacting protein partners is an essential component on a path to understand various cellular functions. Transposon-based systems, particularly those involving in vitro reactions, offer exhaustive insertion mutant libraries and high-throughput platforms for many types of genetic analyses. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision that is based on transposition-assisted construction, sampling, and analysis of a comprehensive insertion mutant library. The methodology integrates random pentapeptide mutagenesis of proteins, yeast two-hybrid screening, and high-resolution genetic footprinting. This straightforward strategy is general, and it provides a rapid and easy means to identify critical contact regions in proteins without the requirement of prior structural knowledge.     

Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, <Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis>

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydration-stress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin superfamily that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTI1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Small molecules that target phosphorylation dependent protein–protein interaction

Protein–protein interaction is one of the key events in the signal transduction pathway. The interaction changes the conformations, activities, localization and stabilities of the proteins, and transduces the signal to the next step. Frequently, this interaction occurs upon the protein phosphorylation. When upstream signals are stimulated, protein kinase(s) is/are activated and phosphorylate(s) their substrates, and induce the phosphorylation dependent protein–protein interaction. For this interaction, several domains in proteins are known to specifically recognize the phosphorylated residues of target proteins. These specific domains for interaction are important in the progression of the diseases caused by disordered signal transduction such as cancer. Thus small molecules that modulate this interaction are attractive lead compounds for the treatment of such diseases. In this review, we focused on three examples of phosphorylation dependent protein–protein interaction modules (14-3-3, polo box domain of Plk1 and F-box proteins in SCF ubiquitin ligases) and summarize small molecules that modulate their interaction. We also introduce our original screening system to identify such small molecules.     

酵母双杂交中诱饵蛋白载体的构建与鉴定方法

酵母双杂交已是研究蛋白质相互作用的经典方法之一。该方法以真核生物酵母为模型,在体内研究活细胞内蛋白质的相互作用,具有高度敏感性,被很多研究者采用。综述了酵母双杂交系统中诱饵蛋白载体的构建,及其在转化酵母中的毒性、自激活性检测研究的最新进展。     

A novel regulatory function of selenocysteine lyase, a unique catalyst to modulate major urinary protein

Mouse selenocysteine lyase (SCL) catalyzes the decomposition of -selenocysteine into -alanine and selenium with pyridoxal 5′-phosphate as a coenzyme. When using SCL as bait in a yeast two-hybrid screening method, major urinary protein I (MUP-I) was identified as a protein that interacts with SCL. This interaction was confirmed with an in vitro binding assay. MUP-I is known as a pheromone-binding protein that accommodates volatile effectors to affect the physiology and behavior of mice. We found that the binding of 2-naphthol to MUP-I was significantly inhibited by SCL, suggesting that SCL regulates the binding capacity of MUP-I.     

Protein interactions of SGP, an essential Streptococcus mutans GTPase, revealed by biochemical and yeast two-hybrid system analyses

SGP, a Streptococcus mutans essential GTPase, plays a role in the stress response of the organism. Recently, we proposed that one of the physiological functions of the SGP is the modulation of the GTP/GDP ratio under different growth conditions. In order to further determine the functions of SGP and its possible interactions with other molecules, we carried out immunoprecipitation, SGP binding, and the yeast two-hybrid system analyses. These approaches suggest that SGP may oligomerize and such interactions could be important for the function of this regulatory protein.     

Identification of Ran-binding protein M as a stanniocalcin 2 interacting protein and implications for androgen receptor activity

Stanniocalcin (STC), a glycoprotein hormone originally discovered in fish, has been implicated in calcium and phosphate homeostasis. While fishes and mammals possess two STC homologs (STC1 and STC2), the physiological roles of STC2 are largely unknown compared with those of STC1. In this study, we identified Ran-binding protein M (RanBPM) as a novel binding partner of STC2 using yeast two-hybrid screening. The interaction between STC2 and RanBPM was confirmed in mammalian cells by immunoprecipitation. STC2 enhanced the RanBPM-mediated transactivation of liganded androgen receptor (AR), but not thyroid receptor β, glucocorticoid receptor, or estrogen receptor β. We also found that AR interacted with RanBPM in both the absence and presence of testosterone (T). Furthermore, we discovered that STC2 recruits RanBPM/AR complex in T-dependent manner. Taken together, our findings suggest that STC2 is a novel RanBPM-interacting protein that promotes AR transactivation. [BMB Reports 2014; 47(11): 643-648]