The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. Overexpression of the mutant SKD1 in cultured mammalian cells caused defect in uptake of transferrin and low-density lipoprotein. This was due to loss of their receptors from the cell surface. The decrease of the surface transferrin receptor (TfR) was correlated with expression levels of the mutant protein. The mutant protein displayed a perinuclear punctate distribution in contrast to a diffuse pattern of the wild-type SKD1. TfR, the lysosomal protein lamp-1, endocytosed dextran, and epidermal growth factor but not markers for the secretory pathway were accumulated in the mutant SKD1-localized compartments. Degradation of epidermal growth factor was inhibited. Electron microscopy revealed that the compartments were exaggerated multivesicular vacuoles with numerous tubulo-vesicular extensions containing TfR and endocytosed horseradish peroxidase. The early endosome antigen EEA1 was also redistributed to these aberrant membranes. Taken together, our findings suggest that SKD1 regulates morphology of endosomes and membrane traffic through them.

Vesicular traffic in eukaryotic cells is characterized by two steps of membrane rearrangement: the formation of vesicles from donor membranes and their fusion with acceptor membranes. With respect to vesicle formation, several of the cytosolic proteins implicated in budding and fission have been identified. A feature common to all these proteins is that their targets, when known, are other proteins rather than lipids. Here we report, using a previously established cell-free system derived from a neuroendocrine cell line, the purification of cytosolic factors that stimulate the formation of constitutive secretory vesicles and immature secretory granules from the trans-Golgi network. One such factor, referred to as CAST1, was identified as the alpha and beta isoforms of the mammalian phosphatidylinositol transfer protein (PtdIns-TP) (refs 3-5). The yeast PtdIns-TP, SEC14p (ref. 6), which has no sequence homology to mammalian PtdIns-TP (refs 7,8), was able to substitute for the mammalian PtdIns-TP in secretory vesicle formation. Our results suggest a highly conserved role for phosphoinositides in vesicle formation.

Monodefluoroborylation of polyfluoroalkenes has been achieved in a regioselective manner under mild conditions via copper catalysis. The method has shown an extremely broad scope of substrates, including (difluorovinyl)arenes, tetrafluoroethylene (TFE), (trifluorovinyl)arenes, and trifluoromethylated monofluoroalkenes. The choice of boron source was important for the efficient transformation of (difluorovinyl)arenes; (Bpin) was suitable for substrates with an electron-deficient aryl group and (Bnep) for those with an electron-rich aryl group. Derivatization of the (fluoroalkenyl)boronic acid esters to the corresponding potassium trifluoroborate salts has rendered the products easily isolable, which greatly improved the synthetic practicality of the monodefluoroborylation reaction. Stoichiometric experiments indicate that the fate of the regioselectivity depends on the mode of β-fluorine elimination, which depends on the substrate. Further transformation of the boryl group has allowed facile preparation of fluoroalkene derivatives as exemplified by the synthesis of a fluoroalkene mimic of atorvastatin, which potently inhibited the enzyme activity of HMG-CoA reductase.

The structural continuity of tight junctions (TJs) is consistently maintained even when epithelial cells divide and move within the cellular sheet. This process is associated with dynamic remodeling of TJs by coordinated internalization and generation of claudin-based TJ strands, but the molecular mechanism behind the regulated turnover of TJs remains largely unknown. In this study, we identified the p80 isoform of the E3 ubiquitin ligase ligand of Numb-protein X1 (LNX1p80) as a protein binding to claudin-1. Interestingly, the concentration of claudins in TJs was remarkably reduced when LNX1p80 was overexpressed in MDCK cells, and there was a reduction not only in the number of TJ strands but also in the amount of detergent-insoluble claudins. We also found that LNX1p80 promoted polyubiquitylation of claudins. This ubiquitylation is dependent on its RING-finger domain and is not mediated by Lys48 of ubiquitin, which is used for protein degradation by the proteasome. Furthermore, LNX1p80 was often colocalized with claudins in vesicular structures containing markers for late endosomes and lysosomes. These findings suggest that LNX1p80 is involved in the ubiquitylation, endocytosis and lysosomal degradation of claudins, and that the turnover of TJs is regulated by ubiquitylation.

Organofluorine compounds are widely used in all aspects of the chemical industry. Although tetrafluoroethylene (TFE) is an example of an economical bulk organofluorine feedstock, the use of TFE is mostly limited to the production of poly(tetrafluoroethylene) and copolymers with other alkenes. Furthermore, no catalytic transformation of TFE that involves carbon-fluorine bond activation has been reported to date. We herein report the first example of a palladium-catalyzed coupling reaction of TFE with arylzinc reagents in the presence of lithium iodide, giving α,β,β-trifluorostyrene derivatives in excellent yields.

In the presence of a Ni(0)/NHC catalyst, an equimolar mixture of aliphatic and aryl aldehydes can be employed to selectively yield a single cross-coupled ester. This reaction can be applied to a variety of aliphatic (1°, 2°, cyc-2°, and 3°) and aryl aldehyde combinations. The reaction represents 100% atom efficiency and generates no waste. Mechanistic studies have revealed that the striking feature of the reaction is the simultaneous coordination of two aldehydes to Ni(0).

Herein, acopper-catalyzed C À Fb ond defluorosilylation reaction of tetrafluoroethylene and other polyfluoroalkenes is described. Mechanistic studies,b ased on as eries of stoichiometric reactions with copper complexes,r evealed that the key steps of this defluorosilylation reaction are 1) the 1,2-addition of as ilylcopper intermediate to the polyfluoroalkene and 2) as ubsequent selective b-fluorine elimination, which generates aC u À Fs pecies.T he b-fluorine elimination is facilitated by Lewis acidic FÀBpin, whichi sg enerated in situ during the defluorosilylation.

A new strategy for C–C bond formation with organoboronates through C–F activation of fluorinated alkenes and arenes was developed. In this Pd‐catalyzed Suzuki–Miyaura‐type cross‐coupling reaction, neither a base for enhancing the reactivity of the organoboron reagents nor a Lewis acid for promoting C–F bond activation was required. A fluoropalladium intermediate played an essential role in this reaction. In addition, a Ni(NHC) catalyst was efficient for C–C coupling through C–F bond activation of fluoroarenes.