Kazuhisa Ota

Novel modular domain PB1 recognizes PC motif to mediate functional protein-protein interactions.

Modular domains mediating specific protein–protein interactions play central roles in the formation of complex regulatory networks to execute various cellular activities. Here we identify a novel domain PB1 in the budding yeast protein Bem1p, which functions in polarity establishment, and mammalian p67phox, which activates the microbicidal phagocyte NADPH oxidase. Each of these specifically recognizes an evolutionarily conserved PC motif to interact directly with Cdc24p (an essential protein for cell polarization) and p40phox (a component of the signaling complex for the oxidase), respectively. Swapping the PB1 domain of Bem1p with that of p67phox, which abolishes its interaction with Cdc24p, confers on cells temperature‐ sensitive growth and a bilateral mating defect. These phenotypes are suppressed by a mutant Cdc24p harboring the PC motif‐containing region of p40phox, which restores the interaction with the altered Bem1p. This domain‐swapping experiment demonstrates that Bem1p function requires interaction with Cdc24p, in which the PB1 domain and the PC motif participate as responsible modules.

Roles for the Two-hybrid System in Exploration of the Yeast Protein Interactome

Comprehensive analysis of protein-protein interactions is a challenging endeavor of functional proteomics and has been best explored in the budding yeast. The yeast protein interactome analysis was achieved first by using the yeast two-hybrid system in a proteome-wide scale and next by large-scale mass spectrometric analysis of affinity-purified protein complexes. While these interaction data have led to a number of novel findings and the emergence of a single huge network containing thousands of proteins, they suffer many false signals and fall short of grasping the entire interactome. Thus, continuous efforts are necessary in both bioinformatics and experimentation to fully exploit these data and to proceed another step forward to the goal. Computational tools to integrate existing biological knowledge buried in literature and various functional genomic data with the interactome data are required for biological interpretation of the huge protein interaction network. Novel experimental methods have to be developed to detect weak, transient interactions involving low abundance proteins as well as to obtain clues to the biological role for each interaction. Since the yeast two-hybrid system can be used for the mapping of the interaction domains and the isolation of interaction-defective mutants, it would serve as a technical basis for the latter purpose, thereby playing another important role in the next phase of protein interactome research.

A functional single-nucleotide polymorphism in the human cytidine deaminase gene contributing to ara-C sensitivity.

To test the hypothesis that analyses of drug targets for polymorphism will help to establish gene-based information for the treatment of cancer patients, we investigated the functional single-nucleotide polymorphisms in the human cytidine deaminase (HDCA) gene. The cDNAs from 52 leukaemia/lymphoma samples and 169 control blood samples were direct-sequenced and analysed for the polymorphisms. Three different polymorphisms (A79C, G208A and T435C) were identified in the coding region of the HDCA gene and displayed allelic frequencies of 20.1%, 4.3% and 70.1%, respectively. No association with susceptibility to disease was observed. A novel polymorphism, G208A produced an alanine to threonine substitution (A70T) within the conserved catalytic domain. By introduction of the polymorphic HCDA genes into the yeast CDA-null mutants, the HCDA-70T showed 40% and 32% activity of prototype for cytidine and ara-C substrates, respectively (P < 0.01). The ara-C IC50 value of the yeast transformants carrying HCDA-70T was 757 +/- 33 micromol and was significantly lower (P < 0.01) than that of prototype (941 +/- 58 micromol). This study demonstrated a population characterized with 208A genotype for, which potentially leads one more sensitive to ara-C treatment than prototype. Accumulation of polymorphisms in the genes responsible for drug metabolism and determination of polymorphism-induced biological variations could provide the additional therapeutic strategies in risk-stratified protocols for the treatment of childhood malignancies.

Molecular Recognition in Dimerization between PB1 Domains

The PB1 (Phox and Bem 1) domain is a recently identified module that mediates formation of a heterodimeric complex with other PB1 domain, e.g. the complexes between the phagocyte oxidase activators p67phox and p40phox and between the yeast polarity proteins Bem1p and Cdc24p. These PB1 domains harbor either a conserved lysine residue on one side or an acidic OPCA (OPR/PC/AID) motif around the other side; the lysine of p67phox or Bem1p likely binds to the OPCA of p40phox or Cdc24p, respectively, via electrostatic interactions. To further understand molecular recognition by PB1 domains, here we investigate the interactions mediated by proteins presenting both the lysine and OPCA on a single PB1 domain, namely Par6, atypical protein kinase C (aPKC), and ZIP. Par6 and aPKC form a complex via the interaction of the Par6 lysine with aPKC-OPCA but not via that between the aPKC lysine and Par6-OPCA, thereby localizing to the tight junction of epithelial cells. aPKC also uses its OPCA to interact with ZIP, another protein that has a PB1 domain presenting both the lysine and OPCA, whereas aPKC binds via the conserved lysine to MEK5 in the same manner as ZIP interacts with MEK5. In addition, ZIP can form a homotypic complex via the conserved electrostatic interactions. Thus the PB1 domain appears to be a protein module that fully exploits its two mutually interacting elements in molecular recognition to expand its repertoire of protein-protein interactions.

A novel Cdc42-interacting domain of the yeast polarity establishment protein Bem1. Implications for modulation of mating pheromone signaling.

In Saccharomyces cerevisiae, the Rho-type small GTPase Cdc42 is activated by its guanine-nucleotide exchange factor Cdc24 to polarize the cell for budding and mating. A multidomain protein Bem1 interacts not only with Cdc42 but also with Cdc24 and the effectors of Cdc42, including the p21-activated kinase Ste20, to function as a scaffold for cell polarity establishment. Although Bem1 interacts with Cdc24 and Ste20 via its PB1 and the second SH3 domains (SH3b), respectively, it is unclear how Bem1 binds Cdc42. Here we show that a region comprising the SH3b and its C-terminal flanking segment termed CI (SH3b-CI) directly interacts with Cdc42. A dual-bait reverse two-hybrid approach revealed that the CI is critical to the interaction: N253D substitution in the CI abolishes the binding of the SH3b-CI to Cdc42 but not to the proline-rich region of Ste20, whereas W192K substitution in the SH3b has the opposite effect. Nevertheless, the SH3b-CI interacts with Ste20 proline-rich region and Cdc42 in a mutually exclusive manner. The N253D substitution renders cellular growth temperature-sensitive and suppresses mating. The W192K-induced mating defect is exacerbated by the N253D substitution and suppressed by increasing the dosage of Ste20 provided that the CI is intact. Intriguingly, Cdc42 can mediate an indirect interaction of the SH3b-CI to the CRIB domain of Ste20. These results suggest that the SH3b and the CI collaborate in tethering of Ste20 to Bem1 to ensure efficient mating pheromone signaling.

Circular permutation of ligand‐binding module improves dynamic range of genetically encoded FRET‐based nanosensor

Quantitative measurement of small molecules with high spatiotemporal resolution provides a solid basis for correct understanding and accurate modeling of metabolic regulation. A promising approach toward this goal is the FLIP (fluorescent indicator protein) nanosensor based on bacterial periplasmic binding proteins (PBPs) and fluorescence resonance energy transfer (FRET) between the yellow and cyan variants of green fluorescent protein (GFP). Each FLIP has a PBP module that specifically binds its ligand to induce a conformation change, leading to a change in FRET between the two GFP variant modules attached to the N‐ and C‐termini of the PBP. The larger is the dynamic range the more reliable is the measurement. Thus, we attempted to expand the dynamic range of FLIP by introducing a circular permutation with a hinge loop deletion to the PBP module. All the six circularly permutated PBPs tested, including structurally distinct Type I and Type II PBPs, showed larger dynamic ranges than their respective native forms when used for FLIP. Notably, the circular permutation made three PBPs, which totally failed to show FRET change when used as their native forms, fully capable of functioning as a ligand binding module of FLIP. These FLIPs were successfully used for the determination of amino acid concentration in complex solutions as well as real‐time measurement of amino acid influx in living yeast cells. Thus, the circular permutation strategy would not only improve the performance of each nanosensor but also expand the repertoire of metabolites that can be measured by the FLIP nanosensor technology.

A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p

Ubiquitination plays various critical roles in eukaryotic cellular regulation and is medated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1–Cullin–F‐box protein complex comprises the largest E3 family, in each member of which a unique F‐box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F‐box proteins, most of them have remained as “orphans” because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC‐PAP‐MS approach to compare ubiquitinated proteins between yeast cells with and without over‐expressed Mdm30p, an F‐box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination‐mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

The yeast eIF4E-associated protein Eap1p attenuates GCN4 translation upon TOR-inactivation.

Amino acid‐starved yeast activates the eIF2α kinase Gcn2p to suppress general translation and to selectively derepress the transcription factor Gcn4p, which induces various biosynthetic genes to elicit general amino acid control (GAAC). Well‐fed yeast activates the target of rapamycin (TOR) to stimulate translation via the eIF4F complex. A crosstalk was demonstrated between the pathways for GAAC and TOR signaling: the TOR‐specific inhibitor rapamycin activates Gcn2p. Here we demonstrate that, upon TOR‐inactivation, the putative TOR‐regulated eIF4E‐associated protein Eap1p likely functions downstream of Gcn2p to attenuate GCN4 translation via a mechanism independent of eIF4E‐binding, thereby constituting another interface between the two pathways.

A parallel affinity purification method for selective isolation of polyubiquitinated proteins.

We developed a parallel affinity purification (PAP) procedure, in which ubiquitinated proteins are purified from the cells that coexpress two affinity‐tagged ubiquitins by sequential use of affinity chromatography specific to each tag. In contrast with previous procedures using a single affinity‐tagged ubiquitin, the PAP eliminates highly abundant ubiquitin monomers and monoubiquitinated proteins to selectively enrich proteins bearing both affinity‐tags, or poly‐ and multiubiquitinated proteins. Accordingly, it would serve as a powerful method to facilitate mass‐spectrometric identification of ubiquitinated proteins.

Remodeling of the SCF complex-mediated ubiquitination system by compositional alteration of incorporated F-box proteins

Ubiquitination regulates not only the stability but the localization and activity of substrate proteins involved in a plethora of cellular processes. The Skp1–Cullin–F‐box protein (SCF) complexes constitute a major family of ubiquitin protein ligases, in each member of which an F‐box protein serves as the variable component responsible for substrate recognition, thereby defining the function of each complex. Here we studied whether the composition of F‐box proteins in the SCF complexes is remodeled under different conditions. We exploited stable isotope labeling and MS for relative quantification of F‐box proteins in the SCF complexes affinity‐purified en masse from budding yeast cells at log and post‐diauxic phases, and revealed an increment of Saf1, an F‐box protein involved in entry into quiescence, during the diauxic shift. Similarly, we found that Met4 overexpression induces a specific increment of Met30, the F‐box protein responsible for ubiquitination of Met4. These results illustrate a cellular response to environmental and genetic perturbations through remodeling of the SCF complex‐mediated ubiquitination system. Compositional alteration of incorporated F‐box proteins may redirect the activity of this system toward appropriate substrates to be ubiquitinated under individual conditions for the maintenance of cellular homeostasis.