Senye Takahashi

Rab11 regulates exocytosis of recycling vesicles at the plasma membrane

Summary Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We show here that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of Sec15, which interacts with Rab11, or Exo70, both components of the exocyst tethering complex, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

A Second Mutant Allele of Furin in the Processing-incompetent Cell Line, LoVo EVIDENCE FOR INVOLVEMENT OF THE HOMO B DOMAIN IN AUTOCATALYTIC ACTIVATION

Furin is a Golgi membrane-associated endoprotease that is involved in cleavage of various precursor proteins predominantly at Arg-X-Lys/Arg-Arg sites. Furin itself is synthesized as an inactive precursor, which is activated through intramolecular autocatalytic cleavage at an Arg-X-Lys-Arg site. We previously found that human colon carcinoma LoVo cells have a frameshift mutation within the homo B domain of furin and thereby lack processing activity toward Arg-X-Lys/Arg-Arg sites. In this study, however, we identified a second furin mutation in this cell line. The mutation, a replacement of a conserved Trp residue within the homo B domain with Arg, results in lack of processing activity of the mutant furin. The combination of both mutations can account for the recessive nature of the processing incompetence of LoVo cells. Immunofluorescence analysis with three distinct anti-furin monoclonal antibodies revealed that neither furin mutant underwent the autocatalytic activation or left the endoplasmic reticulum for the Golgi. These data indicate that the homo B domain as well as the catalytic domain is required for autocatalytic activation of furin.

The E3 ubiquitin ligase LNX1p80 promotes the removal of claudins from tight junctions in MDCK cells.

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.

Functional Cross-Talk between Rab14 and Rab4 through a Dual Effector, RUFY1/Rabip4

Rab14 binds in a GTP-dependent manner to RUFY1/Rabip4, which had been originally identified as a Rab4 effector. We suggest that Rab14 and Rab4 act sequentially; Rab14 is required for recruitment of RUFY1 onto endosomes and subsequent RUFY1 interaction with Rab4 may allow endosomal tethering and fusion.

Structural basis for Arf6-MKLP1 complex formation on the Flemming body responsible for cytokinesis.

A small GTPase, Arf6, is involved in cytokinesis by localizing to the Flemming body (the midbody). However, it remains unknown how Arf6 contributes to cytokinesis. Here, we demonstrate that Arf6 directly interacts with mitotic kinesin‐like protein 1 (MKLP1), a Flemming body‐localizing protein essential for cytokinesis. The crystal structure of the Arf6–MKLP1 complex reveals that MKLP1 forms a homodimer flanked by two Arf6 molecules, forming a 2:2 heterotetramer containing an extended β‐sheet composed of 22 β‐strands that spans the entire heterotetramer, suitable for interaction with a concave membrane surface at the cleavage furrow. We show that, during cytokinesis, Arf6 is first accumulated around the cleavage furrow and, prior to abscission, recruited onto the Flemming body via interaction with MKLP1. We also show by structure‐based mutagenesis and siRNA‐mediated knockdowns that the complex formation is required for completion of cytokinesis. A model based on these results suggests that the Arf6–MKLP1 complex plays a crucial role in cytokinesis by connecting the microtubule bundle and membranes at the cleavage plane.

Distinct roles of Rab11 and Arf6 in the regulation of Rab11-FIP3/arfophilin-1 localization in mitotic cells

Rab11 family interacting protein 3/arfophilin‐1 is a dual effector of Rab11 and Arf6 and exhibits Rab11‐dependent localization to recycling endosomes in interphase. Furthermore, FIP3 undergoes dynamic redistribution to the intercellular bridge during cytokinesis. However, regulation of FIP3 redistribution and its local function by Rab11 and Arf6 has remained controversial. In this study, we developed a procedure for detecting endogenous FIP3, Arf6, and Rab11 and determined that FIP3 is localized near the intercellular bridge during cytokinesis, and to the Flemming body (the midbody) immediately before abscission; Rab11 is localized near the intercellular bridge, but not to the Flemming body; and Arf6 is localized to the Flemming body. Time‐lapse analyses showed that FIP3 is transported to the intercellular bridge during cytokinesis, together with Rab11; before abscission, FIP3 becomes localized to the Flemming body, where Arf6 is already present. After abscission, FIP3 and Arf6 are incorporated into one of the daughter cells as a Flemming body remnant. Based on these observations, we propose that FIP3 localization to recycling endosomes in interphase and their transport to the intercellular bridge during cytokinesis depend on Rab11, and targeting of FIP3‐positive endosomal vesicles to the Flemming body in the abscission phase depends on Arf6.

Regulation of BiP Gene Expression by Cyclopentenone Prostaglandins through Unfolded Protein Response Element

Δ12-Prostaglandin (PG) J2, a cyclopentenone prostaglandin, plays a role in various stress responses. BiP, a stress-inducible chaperone protein, is implicated in protein folding and translocation in endoplasmic reticulum and induced in the condition of accumulation of unfolded proteins. Here, we examined the effect of Δ12-PGJ2 on the expression of the BiP gene. Δ12-PGJ2 markedly stimulated the expression of the BiP gene in a time- and concentration-dependent manner in HeLa cells. This stimulation was specific for cyclopentenone PGs among various PGs. Cycloheximide pretreatment completely inhibited the Δ12-PGJ2-induced expression of the BiP gene, suggesting that the effects on nascent protein synthesis are involved in the signaling mechanism. Δ12-PGJ2 markedly stimulated the promoter activity of the 5′-flanking region of the BiP gene through the unfolded protein response element. Furthermore, Δ12-PGJ2 stimulated the enhancer activity of the 3′-half of the unfolded protein response element, and this stimulation required three nucleotides within this region. Gel mobility shift assay demonstrated that this region was occupied with two specific nuclear protein factors with different mobilities in the control cells, and Δ12-PGJ2 induced the dissociation of the protein-DNA complex with lower mobility. These findings indicate that Δ12-PGJ2 stimulates the expression of BiP gene through the 3′-half of the unfolded protein response element.

Involvement of protein kinase in Δ12-prostaglandin J2-induced expression of rat heme oxygenase-1 gene

We recently identified the cis‐regulatory element and its specific nuclear binding factors for Δ 12‐prostaglandin (PG) J2‐induced expression of the rat heme oxygenase, HO‐1 [Koizumi, T., Odani, N., Okuyama, T., Ichikawa, A. and Negishi, M. (1995)J. Biol. Chem. 270, in press]. Here we further examined the molecular mechanism underlying the Δ 12‐PGJ2‐induced HO‐1 gene expression. Protein kinase inhibitors, 2‐aminopurine and staurosporine, suppressed the Δ 12‐PGJ2‐induced HO‐1 mRNA and the nuclear protein binding to the Δ 12‐PGJ2‐responsivecis‐regulatory element in rat basophilic leukemia cells. Furthermore, the nuclear protein binding to the element was suppressed by in vitro phosphatase treatment of the nuclear proteins from Δ 12‐PGJ2‐treated cells. These findings suggest that Δ 12‐PGJ2 induces the expression of the HO‐1 gene through phosphorylation of the nuclear proteins which bind to the Δ 12‐PGJ2‐responsive element.

Regulation of ciliary retrograde protein trafficking by the Joubert syndrome proteins ARL13B and INPP5E

ABSTRACT ARL13B (a small GTPase) and INPP5E (a phosphoinositide 5-phosphatase) are ciliary proteins encoded by causative genes of Joubert syndrome. We here showed, by taking advantage of a visible immunoprecipitation assay, that ARL13B interacts with the IFT46–IFT56 (IFT56 is also known as TTC26) dimer of the intraflagellar transport (IFT)-B complex, which mediates anterograde ciliary protein trafficking. However, the ciliary localization of ARL13B was found to be independent of its interaction with IFT-B, but dependent on the ciliary-targeting sequence RVEP in its C-terminal region. ARL13B-knockout cells had shorter cilia than control cells and exhibited aberrant localization of ciliary proteins, including INPP5E. In particular, in ARL13B-knockout cells, the IFT-A and IFT-B complexes accumulated at ciliary tips, and GPR161 (a negative regulator of Hedgehog signaling) could not exit cilia in response to stimulation with Smoothened agonist. This abnormal phenotype was rescued by the exogenous expression of wild-type ARL13B, as well as by its mutant defective in the interaction with IFT-B, but not by its mutants defective in INPP5E binding or in ciliary localization. Thus, ARL13B regulates IFT-A-mediated retrograde protein trafficking within cilia through its interaction with INPP5E. Summary: Mutations in the genes encoding ARL13B and INPP5E are causative for Joubert syndrome. ARL13B interacts with INPP5E and regulates IFT-A-mediated retrograde protein trafficking within cilia.

Modification of the N-terminus of membrane fusion-active peptides blocks the fusion activity.

The amphiphilic anionic peptides E5 and E5L can mimic the fusogenic activity of influenza hemagglutinin(HA). These peptides induced fusion of egg yolk phosphatidylcholine small or large unilamellar vesicles only at acidic pH in a similar manner to viral HA. Acetylation or acetimidylation of the N-terminus of the peptides drastically reduced the fusion activity of the intact peptides, while C-terminal amidation left the activity unchanged. The binding assay suggested that the interaction of the modified peptides with lipid membranes was almost unchanged in comparison with those of the parent peptides, and the CD spectra showed that these peptides were alpha-helical. The results showed the importance of the N-terminus of the peptides on the membrane fusion activity, although why the N-terminal modifications affect the activity is still unclear.