PDZD8-FKBP8 tethering complex at ER-mitochondria contact sites regulates mitochondrial complexity.

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

Comparison of the conventional and immersing powdered crude drugs (IPCD) methods for color and extraction of quantitative indicator ingredients in the Kampo formula decoction of daiokanzoto.

AIM: The immersing powdered crude drugs (IPCD) method is a quick and simple method for preparing decoctions. Here, the conventional and IPCD methods were compared for the color and extraction of quantitative indicator ingredients in the daiokanzoto decoction solution, and the suitability of the IPCD method was assessed. METHODS: The color of decoction solutions was visually observed, and the Commission Internationale de L'éclairage (CIE) L*a*b*color parameters were measured using conventional and IPCD methods. The extracted amounts of sennoside A and glycyrrhizic acid, which are quantitative indicator ingredients of rhubarb and glycyrrhiza, respectively, were quantified. RESULTS: Using both methods, the decoction solution colors were strong for rhubarb alone and daiokanzoto but weak for glycyrrhiza alone. The color change of daiokanzoto was thought to be primarily caused by rhubarb alone. The L*a*b* values of the decoction solution determined by the IPCD method were comparable to those determined by the conventional method (60 min). Using the conventional method, sennoside A and glycyrrhizic acid were mostly extracted in 10 and 30 min, respectively. Using the IPCD method, both sennoside A and glycyrrhizic acid were fully extracted in 2 min. The IPCD method yielded significantly more sennoside A and glycyrrhizic acid (2 times and 1.5 times, respectively) than the conventional method (60 min). CONCLUSION: The IPCD method was found to be comparable to the conventional method in terms of the color, and using IPCD method, the same or greater amounts of quantitative indicator ingredients of crude drugs in the decoction of daiokanzoto compared to the conventional method. It was suggested that there are limitations to assessing the equivalence of decoctions from decoction color. The IPCD method may be a useful method although it is prudent to use the IPCD method for Kampo formula decoction in clinical practice with a certain degree of caution.

Isolation and identification of Zalaria sp. Him3 as a novel fructooligosaccharides-producing yeast.

AIMS: This study aimed at obtaining a novel fructooligosaccharides (FOS)-producing yeast, which was different from conventional FOS producers, Aureobasidium spp. METHODS AND RESULTS: Strain Him3 was newly isolated from a Japanese dried sweet potato as a FOS producer. The strain exhibited yeast-like cells and melanization on the potato dextrose agar medium, and formed very weak pseudomycelia on the yeast extract polypeptone dextrose agar medium. Based on the internal transcribed spacer (ITS) region of ribosomal DNA and a partial ß-tubulin gene sequences, the strain Him3 was identified as Zalaria sp. The ß-fructofuranosidase (FFase) produced by strain Him3 was localized on the cell surface (CS-FFase) as well as in the culture broth (EC-FFase). The FOS production yields by CS-FFase and EC-FFase from 50% sucrose were 63.8% and 64.6%, respectively, to consumed sucrose after the reaction for 72 h. CONCLUSIONS: We successfully isolated a novel black yeast, Zalaria sp. Him3, with effective capacity for FOS production. Phylogenetic analysis revealed that strain Him3 was distantly related with the conventional FOS producers, Aureobasidium spp. SIGNIFICANCE AND IMPACT OF THE STUDY: Since FFase of strain Him3 demonstrated high production yields of FOS, it could be applied to novel industrial production of FOS, which is different from conventional methods.