Sperm–egg fusion is indispensable for completing mammalian fertilization. Although the underlying molecular mechanisms are poorly understood, requirement of two spermatozoon factors, IZUMO1 and SPACA6, and two oocyte factors, CD9 and the IZUMO1 counter-receptor JUNO, has been proven by gene disruption, and the binding of cells to an oocyte can be reconstituted by ectopic expression of IZUMO1. Here we demonstrate that robust IZUMO1-dependent adhesion of sperm with an oocyte accompanies the dimerization of IZUMO1. Despite the intrinsic dimeric property of its N-terminal region, IZUMO1 is monomeric in spermatozoa. Interestingly, JUNO associates with monomeric IZUMO1, which is then quickly removed as tight adhesion of the two cells is subsequently established. We therefore propose that global structural rearrangement of IZUMO1 occurs on JUNO recognition and that this rearrangement may then initiate force generation to overcome repulsion between the juxtaposing membranes, through an unidentified receptor on the egg.

Werner syndrome (WS) is a rare autosmomal recessive genetic disorder causing premature aging. The gene (WRN) responsible for WS encodes a protein homologous to the RecQ-type helicase. WRN has a nucleolar localization signal and shows intranuclear tra cking between the nucleolus and the nucleoplasm. WRN is recruited into the nucleolus when rRNA transcription is reactivated in quiescent cells. Inhibition of mRNA transcription with a-amanitin has no e ect on nucleolar localization of WRN whereas inhibition of rRNA transcription with actinomycin D releases WRN from nucleoli, suggesting that nucleolar WRN is closely related to rRNA transcription by RNA polymerase I (RPI). A possible function of WRN on rRNA transcription through interaction with RPI is supported by the results described here showing that WRN is coimmunoprecipitated with an RPI subunit, RPA40. Here we show that WS ®broblasts are characterized by a decreased level of rRNA transcription compared with wild-type cells, and that the decreased level of rRNA transcription in WS ®broblasts recovers when wild-type WRN is exogenously expressed. By contrast, exogenously expressed mutant-type WRN lacking an ability to migrate into the nucleolus fails to stimulate rRNA transcription. These results suggest that WRN promotes rRNA transcription as a component of an RPIassociated complex in the nucleolus.

The occurrence of a hitherto unknown pathway involving the action of two enzymes, a nitrile hydratase and an amidase for the biosynthesis of indole-3-acetic acid was discovered in phytopathogenic bacteria Agrobacterium tumefaciens and in leguminous bacteria Rhizobium. The nitrile hydratase acting on indole-3-acetonitrile was purified to homogeneity through only two steps from the cell-free extract ofA. tumefaciens. The molecular mass of the purified enzyme estimated by HPLC was about 102 kDa, and the enzyme consisted of four subunits identical in molecular mass. The enzyme exhibited a broad absorption spectrum in the visible range with absorption maxima at 408 nm and 705 nm, and it contained cobalt and iron. The enzyme stoichiometrically catalyzed the hydration of indole-3-acetonitrile into indole-3-acetamide with a specific activity of 13.7 ,umol per min per mg and a Km of 7.9 ,uM.Indole-3-acetic acid (IAA) is a well-known phytohormone and three biosynthetic routes involving indole-3-pyruvate, tryptamine, or indole-3-acetonitrile have been studied in higher plants (1, 2). In the indole-3-acetonitrile route, the nitrile generated from indole-3-acetaldoxime, naturally occurring in plants (3), by indoleacetaldoxime dehydratase (EC 4.2.1.29) (4) or from indole-3-methylglucosinolate by myrosinase (EC 3.2.3.1) (5), is converted into IAA by nitrilase (EC 3.5.5.1) in Brassicaceae (cabbage group and radish), Gramineae (grasses), and Musaceae (banana family) (6, 7). While studies on auxin have been made for >100 years (8), none of the genes encoding IAA-forming enzymes in the indole-3-pyruvate or the tryptamine route have been cloned yet, although cDNA cloning of nitrilases fromArabidopsis thaliana (9-11) has been reported.Microbial production of plant hormones such as IAA and cytokinin seems to be essential for the virulence of bacteria in their host plants (12). Agrobacterium tumefaciens (Ag. tumefaciens) can infect wound sites on a wide range of dicotyledonous plants and cause the formation of crown gall tumors by transfer of the transferred DNA (T-DNA) region in the Ti plasmid from the bacteria into plant cells. The T-DNA genes tms-1 and tms-2 encode tryptophan 2-monooxygenase (EC 1.13.12.3) and indoleacetamide hydrolase, respectively (13); IAA is produced from tryptophan by the sequential action of these two enzymes. A similar situation in the genes responsible for IAA synthesis was observed in Pseudomonas savastanoi (Ps. savastanoi), which induced tissue proliferation in olive and oleander plants (14).In studying nitrile metabolism, we have found that the microbial degradation of nitriles can proceed by two enzymatic pathways (15). In one, a nitrilase catalyzes the direct conversion of nitriles into the corresponding acids plus ammonia (Eq. 1) (16). Yamada and coworkers (17) in our laboratory have purified and characterized such a nitrilase from Alcaligenes faecalis acting on indole-3-acetonitrile, and we have cloned (18) the gene. In the other pathway, a nitrile hydratase catalyzes the hydration of nitrile...

Molecular imaging employing fluorescent proteins has been widely used to highlight specific reactions or processes in various fields of the life sciences. Despite extensive improvements of the fluorescent tag, this technology is still limited in the study of molecular events in the extracellular milieu. This is partly due to the presence of cysteine in the fluorescent proteins. These proteins almost cotranslationally form disulfide bonded oligomers when expressed in the endoplasmic reticulum (ER). Although single molecule photobleaching analysis showed that these oligomers were not fluorescent, the fluorescent monomer form often showed aberrant behavior in folding and motion, particularly when fused to cysteine-containing cargo. Therefore we investigated whether it was possible to eliminate the cysteine without losing the brightness. By site-saturated mutagenesis, we found that the cysteine residues in fluorescent proteins could be replaced with specific alternatives while still retaining their brightness. cf(cysteine-free)SGFP2 showed significantly reduced restriction of free diffusion in the ER and marked improvement of maturation when fused to the prion protein. We further applied this approach to TagRFP family proteins and found a set of mutations that obtains the same level of brightness as the cysteine-containing proteins. The approach used in this study to generate new cysteine-free fluorescent tags should expand the application of molecular imaging to the extracellular milieu and facilitate its usage in medicine and biotechnology.

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (x-1x-3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane-bound native protein. The results are consistent with our notion that the native protein is targeted to the membrane by a post-translational modification mechanism. We also discovered that P450foxy could use shorter saturated fatty acid chains 1 (C9 and C10) as a substrate. The regiospecificity (x-1x-3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates. Steady state kinetic studies showed that the k cat values for all substrates tested (C9-C16) were of the same magnitude (1200-1800 min )1 ), whereas the catalytic efficiency (k cat /K m ) was higher for longer fatty acids. Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length. This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified. Kinetic data were consistent with the inhibition due to binding of the second substrate. We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding.

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Carnivorous arthropods use volatile infochemicals emitted from prey-infested plants in their foraging behavior. Although several volatile components are common among plant species, the compositions differ among prey-plant complexes. Studies showed that the predatory mite Neoseiulus womersleyi is attracted only to previously experienced plant volatiles. In this study, we identified the attractant components in prey-induced plant volatiles of two prey-plant complexes. N. womersleyi reared on Tetranychus kanzawai-infested tea leaves showed significant preference for a mixture of three synthetic compounds [mimics of the T. kanzawai-induced tea leaves volatiles: (E)-beta-ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), and (E,E)-alpha-farnesene] at a level comparable to that for T. kanzawai-induced tea plant volatiles. However, mixtures lacking any of these compounds did not attract the predatory mites. Likewise, N. womersleyi reared on T. urticae-infested kidney bean plants showed a significant preference for a mixture of four synthetic compounds [mimics of the T. urticae-induced kidney bean volatiles: DMNT, methyl salicylate (MeSA), beta-caryophyllene, and (E,E)-4,8,12-trimethyl-1,3,7,11-tridecatetraene] at a level comparable to that for T. urticae-induced kidney bean volatiles. The absence of any of the four compounds resulted in no attraction. These results indicate that N. womersleyi can use at least four volatile components to identify prey-infested plants.