Hiroki Inoue

Ciliary targeting motif VxPx directs assembly of a trafficking module through Arf4

Dysfunctions of primary cilia and cilia‐derived sensory organelles underlie a multitude of human disorders, including retinal degeneration, yet membrane targeting to the cilium remains poorly understood. Here, we show that the newly identified ciliary targeting VxPx motif present in rhodopsin binds the small GTPase Arf4 and regulates its association with the trans‐Golgi network (TGN), which is the site of assembly and function of a ciliary targeting complex. This complex is comprised of two small GTPases, Arf4 and Rab11, the Rab11/Arf effector FIP3, and the Arf GTPase‐activating protein ASAP1. ASAP1 mediates GTP hydrolysis on Arf4 and functions as an Arf4 effector that regulates budding of post‐TGN carriers, along with FIP3 and Rab11. The Arf4 mutant I46D, impaired in ASAP1‐mediated GTP hydrolysis, causes aberrant rhodopsin trafficking and cytoskeletal and morphological defects resulting in retinal degeneration in transgenic animals. As the VxPx motif is present in other ciliary membrane proteins, the Arf4‐based targeting complex is most likely a part of conserved machinery involved in the selection and packaging of the cargo destined for delivery to the cilium.

Decreased extra-renal urate excretion is a common cause of hyperuricemia

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate overproduction type and/or underexcretion type based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the overproduction type in the current concept of hyperuricemia be renamed renal overload type, which consists of two subtypes—extra-renal urate underexcretion and genuine urate overproduction—providing a new concept valuable for the treatment of hyperuricemia and gout.

Arf GAPs and their interacting proteins.

Membrane trafficking and remodeling of the actin cytoskeleton are critical activities contributing to cellular events that include cell growth, migration and tumor invasion. ADP‐ribosylation factor (Arf)‐directed GTPase activating proteins (GAPs) have crucial roles in these processes. The Arf GAPs function in part by regulating hydrolysis of GTP bound to Arf proteins. The Arf GAPs, which have multiple functional domains, also affect the actin cytoskeleton and membranes by specific interactions with lipids and proteins. A description of these interactions provides insights into the molecular mechanisms by which Arf GAPs regulate physiological and pathological cellular events. Here we describe the Arf GAP family and summarize the currently identified protein interactors in the context of known Arf GAP functions.

Consensus nomenclature for the human ArfGAP domain-containing proteins

At the FASEB summer research conference on “Arf Family GTPases”, held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

Src-Dependent Phosphorylation of ASAP1 Regulates Podosomes

ABSTRACT Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.

Arf GAPs as regulators of the actin cytoskeleton

The Arf (ADP‐ribosylation factor) GAPs (GTPase‐activating proteins) are a family of proteins with a common catalytic domain that induces hydrolysis of GTP bound to Arf GTP‐binding proteins. At least three groups of multidomain Arf GAPs affect the actin cytoskeleton and cellular activities, such as migration and movement, that depend on the cytoskeleton. One role of the Arf GAPs is to regulate membrane remodelling that accompanies actin polymerization. Regulation of membrane remodelling is mediated in part by the regulation of Arf proteins. However, Arf GAPs also regulate actin independently of effects on membranes or Arf. These functions include acting as upstream regulators of Rho family proteins and providing a scaffold for Rho effectors and exchange factors. With multiple functional elements, the Arf GAPs could integrate signals and biochemical activities that result in co‐ordinated changes in actin and membranes necessary for a wide range of cellular functions.

TARGETED DISRUPTION OF THE GENE ENCODING THE PROTEOLIPID SUBUNIT OF MOUSE VACUOLAR H+-ATPASE LEADS TO EARLY EMBRYONIC LETHALITY

Vacuolar H(+)-ATPase (V-ATPase) is responsible for acidification of intracellular compartments in eukaryotic cells. Its 16-kDa subunit (proteolipid, PL16) plays a central role in V-ATPase function, forming the principal channel via which protons are translocated. To elucidate physiological roles of V-ATPase in mammalian cell function and embryogenesis, we attempted to generate a PL16 null mutant mouse by gene-targeting. Mice heterozygous (PL16(+/-)) for the proteolipid mutation were intercrossed and their offspring were classified according to genotype. There were no homozygous (PL16(-/-)) pups among 69 neonates examined, but a few PL16(-/-) embryos were found during the pre-implantation stages of embryonic development, up to day 3.5 post-coitum. These results suggested that PL16 (and hence V-ATPase) may play an essential role in cell proliferation and viability during early embryogenesis. PL16(+/-) mice were indistinguishable from their wild-type littermates and displayed no discernible abnormalities, although the PL16 mRNA level in PL16(+/-) mice decreased to about one-half of wild-type levels.

ABCG2 dysfunction causes hyperuricemia due to both renal urate underexcretion and renal urate overload

Gout is a common disease which results from hyperuricemia. We have reported that the dysfunction of urate exporter ABCG2 is the major cause of renal overload (ROL) hyperuricemia, but its involvement in renal underexcretion (RUE) hyperuricemia, the most prevalent subtype, is not clearly explained so far. In this study, the association analysis with 644 hyperuricemia patients and 1,623 controls in male Japanese revealed that ABCG2 dysfunction significantly increased the risk of RUE hyperuricemia as well as overall and ROL hyperuricemia, according to the severity of impairment. ABCG2 dysfunction caused renal urate underexcretion and induced hyperuricemia even if the renal urate overload was not remarkable. These results show that ABCG2 plays physiologically important roles in both renal and extra-renal urate excretion mechanisms. Our findings indicate the importance of ABCG2 as a promising therapeutic and screening target of hyperuricemia and gout.

A Role for the Ancient SNARE Syntaxin 17 in Regulating Mitochondrial Division

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.

A Novel Kinesin-Like Protein, KIF1Bβ3 Is Involved in the Movement of Lysosomes to the Cell Periphery in Non-Neuronal Cells

The kinesin superfamily protein, KIF1Bβ, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non‐neuronal tissues. To elucidate the functions of KIF1Bβ in non‐neuronal cells, we analyzed the intracellular localization of KIF1Bβ and characterized its isoform expression profile. In COS‐7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bβ, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin‐like protein, KIF1Bβ3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bβ‐specific tail region. Overexpression of KIF1Bβ3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bβ3‐Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot‐Marie‐Tooth disease type 2u2003A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bβ3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.