Structure of human lysosomal membrane glycoprotein 1. Assignment of disulfide bonds and visualization of its domain arrangement

The amino acid sequence of one of the major lysosomal membrane glycoproteins, lysosome-associated membrane protein 1 (lamp-1), was deduced from its cDNA sequence (Fukuda, M., Viitala, J., Matteson, J., and Carlsson, S. R. (1988) J. Biol. Chem. 263, 18920-18928). This amino acid sequence suggests that lamp-1 contains a hinge-like structure and could form disulfide bridges that are observed in the immunoglobulin superfamily. To test this possibility, we have determined the positions of the disulfide bridges by isolating and sequencing cystine-containing peptides which contain disulfide bridges. The results indicate that disulfide arrangement of lamp-1 is different from that of immunoglobulins. Each molecule contains, in total, four loops formed by disulfide bonds, and each loop contains 36-39 amino acid residues. However, none of the disulfide bonds connects two domains that are separated by a hinge-like structure. The results indicate that the hinge region has no ordered structure, and the relative positions of the two domains can be altered in space. Examination of the ultrastructure of lamp-1 by electron microscopy showed that the hinge-like structure actually functions as a hinge. These results indicate that the lamp-1 molecule represents a novel family of glycoproteins with unique structural properties.     

Construction of Yeast Strains with High Cell Surface Lipase Activity by Using Novel Display Systems Based on the Flo1p Flocculation Functional Domain

We constructed a novel cell-surface display system, using as a new type of cell-wall anchor 3,297 or 4,341 bp of the 3′ region of the FLO1 gene (FS or FL gene, respectively), which encodes the flocculation functional domain of Flo1p. In this system, the N terminus of the target protein was fused to the FS or FL protein and the fusion proteins were expressed under the control of the inducible promoter UPR-ICL (5′ upstream region of the isocitrate lyase of Candida tropicalis). Using this new system, recombinant lipase with a pro sequence from Rhizopus oryzae (rProROL), which has its active site near the C terminus, was displayed on the cell surface. Cell-surface display of the FSProROL and FLProROL fusion proteins was confirmed by immunofluorescence microscopy and immunoblotting. Lipase activity reached 145 IU/liter (61.3 IU/g [dry cell weight]) on the surface of the yeast cells, which successfully catalyzed the methanolysis reaction. Using these whole-cell biocatalysts, methylesters synthesized from triglyceride and methanol reached 78.3% after 72 h of reaction. To our knowledge, this is the first example of cell-surface display of lipase with high activity. Interestingly, the yeast cells displaying the FLProROL protein showed strong flocculation, even though the glycosylphosphatidylinositol anchor attachment signal and cell-membrane-anchoring region of Flo1p had been deleted from this gene. The cell-surface display system based on FL thus endows the yeast strain with both novel enzyme display and strong flocculation ability.     

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.     

Distinct responses of cones and melanopsin-expressing retinal ganglion cells in the human electroretinogram

ABSTRACT: BACKGROUND: The discovery of the novel photoreceptor, melanopsin-expressing retinal ganglion cells (mRGCs), has raised researchers' interest in photoreceptive tasks performed by the mRGC, especially in non-image-forming visual functions. In a prior study, we investigated the mRGC response to light stimuli independent of rods and cones with the four-primary illumination system, which modulates stimulus levels to the mRGC and cones independently, and mRGC baseline responses were recorded in the electroretinogram (ERG). METHODS: In the present study, we used the same illumination system to compare independent responses of the mRGC and cones in five subjects (mean +/- SD age, 23.0 +/- 1.7 years). The ERG waveforms were examined as direct measurements of responses of the mRGCs and cones to stimulation (250 msec). Implicit times (the time taken to peaks) and peak values from 30 stimuli given to each subject were analyzed. RESULTS: Two distinct positive peaks appeared in the mRGC response, approximately 80 msec after the onset of the stimuli and 30 msec after their offset, while no such peaks appeared in the cone response. The response to the mRGC stimulus was significantly higher than that to the cone stimulus at ~80 msec (p < 0.05) and tended to be higher than the cone stimulus at ~280 msec (p = 0.08). CONCLUSIONS: Implicit time of the first peak was much longer than that to the b-wave and this delay might reflect mRGC's sluggish responses. This is the first report of amplitudes and implicit time in the ERG from the response of the mRGC that is independent of rods and cones and obtained using the four-primary illumination system.     

Efficient establishment of pure cultures of yellow chanterelle Cantharellus anzutake from ectomycorrhizal root tips,and morphological characteristics of ectomycorrhizae and cultured mycelium

Species of fleshy yellow Cantharellus are known as chanterelles, which are among the most popular wild edible mycorrhizal mushrooms in the world. However, pure culture isolates of Cantharellus are rare. We report an efficient isolation technique of the Japanese golden chanterelle, Cantharellus anzutake, from its ectomycorrhizal root tips. Field-sampled fresh ectomycorrhizal root tips of C. anzutake on various hosts such as pines, spruce, and oaks were vortexed with 0.005% Tween 80 solution, surface sterilized with 1% calcium hypochlorite solution, rinsed with sterilized distilled water, and placed on modified Norkrans’ C (MNC) agar plate medium. Most ectomycorrhizal root tips of C. anzutake produced yellowish mycelial colonies within a few months. In contrast, tissue isolation from basidiomata provided limited cultures of C. anzutake but much contamination of bacteria and molds, even on media that contained antibiotics. The established C. anzutake cultures had clamp connections on the hyphae and contained intracellular oily droplets. These cultured isolates were identified as C. anzutake by sequence analysis of the rRNA internal transcribed spacer (ITS) region and translation elongation factor EF1-alpha (tef-1) genes.     

Root-tip diameters of woody species in subalpine Abies forest

We clarified the differences in root-tip diameter (RTD) among tree and shrub species in an Abies forest. To evaluate the effects of sampling month and tree size on RTD, we measured the root-tip diameters of mature individuals of nine woody species and sapling individuals of two Abies species in a subalpine Abies forest on Mount Shimagare in central Japan. Species, sampling month, and their interaction affected RTD; however, the differences in RTD between some pairs of species were consistent across sampling months. The woody species fell into two groups, based on RTD size: tree species with larger RTDs (group 1) and shrub species with smaller RTDs (group 2). Seasonal changes in RTD were observed in three species and showed different patterns among species. Tree size did not affect RTD for either Abies species; however, there was an interaction between tree size and sampling month for Abies veitchii. The woody species category had the greatest effect on RTD, followed by sampling month and then tree size.     

A possible role of Atg8 homologs as a scaffold for signal transduction

Atg8 and its homologs are essential for autophagosome formation in various species. In animal cells, Atg8 homologs have an additional function in clearance of damaged organelles and bacteria, acting as a landmark for selective autophagy. We have recently shown that OATL1, a Rab-GTPase-activating protein (Rab-GAP), is a novel binding partner of Atg8 homologs in mammalian cells, but to our surprise, it is not a substrate of autophagy. Further analysis indicates that OATL1 is involved in the fusion between autophagosomes and lysosomes through its GAP activity and its Atg8 homolog binding activity. Our findings suggest a novel function of Atg8 homologs as a scaffold for signal transduction that regulates autophagosomal maturation.     

Yeast whole-cell biocatalyst constructed by intracellular overproduction of Rhizopus oryzae lipase is applicable to biodiesel fuel production

Yeast whole-cell biocatalysts for lipase-catalyzed reactions were constructed by intracellularly overproducing Rhizopus oryzae lipase (ROL) in Saccharomvces cerevisiae MT8-1. The gene encoding lipase from R. orvzae IFO4697 was cloned, and intracellular overproduction systems of a recombinant ROL with a pro-sequence (rProROL) were constructed. When rProROL from R. oryzae IFO4697 was produced under the control of the 5'-upstream region of the isocitrate lyase gene of Candida tropicalis (UPR-ICL) at 30 degrees C for 98 h by two-stage cultivation using SDC medium (SD medium with 2% casamino acids) containing 2.0% and 0.5% glucose, intracellular lipase activity reached levels up to 474.5 IU/l. These whole-cell biocatalysts were permeabilized by air-drying and used for the synthesis of methyl esters (MEs), a potential biodiesel fuel, from plant oil and methanol in a solvent-free and water-containing system. The ME content in the reaction mixture was 71 wt% after a 165-h reaction at 37 degrres C with stepwise addition of methanol. These results indicate that an efficient whole-cell biocatalyst can be prepared by intracellular overproduction of lipase in yeast cells and their permeabilization.