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Insulin is an essential peptide hormone that maintains blood glucose levels. Although the mechanisms underlying insulin exocytosis have been investigated, the mechanism of proinsulin export from the endoplasmic reticulum (ER) remains unclear. Here, we demonstrated that Surf4, a cargo receptor homolog, regulates the ER export of proinsulin via its recruitment to ER exit sites (ERES). Under high-glucose conditions, Surf4 expression was upregulated, and Surf4 proteins mainly localized to the ER at a steady state and accumulated in the ERES, along with proinsulin in rat insulinoma INS-1 cells. Surf4-knockdown resulted in proinsulin retention in the ER and decreased the levels of mature insulin in secretory granules, thereby significantly reducing insulin secretion. Surf4 forms an oligomer and can physically interact with proinsulin and Sec12, essential for COPII vesicle formation. Our findings suggest that Surf4 interacts with proinsulin and delivers it into COPII vesicles for ER export in co-operation with Sec12 and COPII.

Proteins synthesized within the endoplasmic reticulum (ER) are exported from ER exit sites via coat protein complex II (COPII)-coated vesicles. Although the mechanisms of COPII-vesicle formation at the ER exit sites are highly conserved among species, vertebrate cells secrete a wide range of materials, including collagens and chylomicrons, which form bulky structures within the ER that are too large to fit into conventional carriers. Transport ANd Golgi Organization 1 (TANGO1) was initially identified as a cargo receptor for collagens but has been recently rediscovered as an organizer of ER exit sites. We would like to review recent advances in the mechanism of large cargo secretion and organization of ER exit sites through the function of TANGO1.

Stimulator of interferon genes (STING) is essential for the type I interferon and pro-inflammatory responses against DNA pathogens. In response to the presence of cytosolic DNA, STING translocates from the endoplasmic reticulum (ER) to the Golgi, and activates TANK-binding kinase 1 (TBK1), a cytosolic kinase that is essential for the activation of STING-dependent downstream signalling. The organelles where TBK1 binds to STING remain unknown. Here we show that TBK1 binds to STING at the Golgi, not at the ER. Treatment with brefeldin A, an agent to block ER-to-Golgi traffic, or knockdown of Sar1, a small GTPase that regulates coat protein complex II (COP-II)-mediated ER-to-Golgi traffic, inhibited the binding of TBK1 to STING. Endogenous TBK1 was recruited to the Golgi when STING was transported to the Golgi, as shown by immunofluorescence microscopy. STING variants that constitutively induce the type I interferon response were found in patients with autoinflammatory diseases. Even these disease-causative STING variants could not bind to TBK1 when the STING variants were trapped in the ER. These results demonstrate that the Golgi is an organelle at which STING recruits and activates TBK1 for triggering the STING-dependent type I interferon response.

Cargo proteins exported from the endoplasmic reticulum to the Golgi apparatus are typically transported in coat protein complex II (COPII)-coated vesicles of 60–90 nm diameter. Several cargo molecules including collagens and chylomicrons form structures that are too large to be accommodated by these vesicles, but their secretion still requires COPII proteins. Here, we first review recent progress on large cargo secretions derived especially from animal models and human diseases, which indicate the importance of COPII proteins. We then discuss the recent isolation of specialized factors that modulate the process of COPII-dependent cargo formation to facilitate the exit of large-sized cargoes from the endoplasmic reticulum. Based on these findings, we propose a model that describes the importance of the GTPase cycle for secretion of oversized cargoes. Next, we summarize reports that describe the structures of COPII proteins and how these results provide insight into the mechanism of assembly of the large cargo carriers. Finally, we discuss what issues remain to be solved in the future.

Hepatic fibrosis is caused by exaggerated wound healing response to chronic injury, which eventually leads to hepatic cirrhosis. Differentiation of hepatic stellate cells (HSCs) to myofibroblast-like cells by inflammatory cytokines is the critical step in fibrosis. This step is accompanied by enlargement of the endoplasmic reticulum (ER) and Golgi apparatus, suggesting that protein synthesis and secretion are augmented in the activated HSCs. However, the process of rearrangement of secretory organelles and their functions remain to be fully elucidated. Here, we revealed that differentiation alters early secretory gene expression. We observed significant isoform-specific upregulation of the inner coat protein complex II (COPII) components, Sec23A and Sec24D, via the transmembrane bZIP transcription factor, CREB3L2/BBF2H7, during HSC activation. Moreover, knockdown of these components abrogated the activation, suggesting that Sec23A/Sec24D-mediated ER to Golgi trafficking is required for HSC activation.

By interacting with the collagen cargo receptor component cTAGE5, Sec12 concentrates at ER exit sites and generates the high levels of GTP-bound Sar1 necessary for export of collagen to the Golgi., Mechanisms for exporting variably sized cargo from the endoplasmic reticulum (ER) using the same machinery remain poorly understood. COPII-coated vesicles, which transport secretory proteins from the ER to the Golgi apparatus, are typically 60–90 nm in diameter. However, collagen, which forms a trimeric structure that is too large to be accommodated by conventional transport vesicles, is also known to be secreted via a COPII-dependent process. In this paper, we show that Sec12, a guanine-nucleotide exchange factor for Sar1 guanosine triphosphatase, is concentrated at ER exit sites and that this concentration of Sec12 is specifically required for the secretion of collagen VII but not other proteins. Furthermore, Sec12 recruitment to ER exit sites is organized by its direct interaction with cTAGE5, a previously characterized collagen cargo receptor component, which functions together with TANGO1 at ER exit sites. These findings suggest that the export of large cargo requires high levels of guanosine triphosphate–bound Sar1 generated by Sec12 localized at ER exit sites.

The engulfment and clearance of apoptotic cells by neighboring cells or professional phagocytes is crucial to tissue homeostasis and the regulation of immune responses. The Arf-like GTPase Arl8, which localizes primarily to lysosomes, mediates phagolysosome formation to promote the efficient degradation of apoptotic germ cells in Caenorhabditis elegans., Efficient clearance of apoptotic cells by phagocytes is important for development, tissue homeostasis, and the prevention of autoimmune responses. Phagosomes containing apoptotic cells undergo acidification and mature from Rab5-positive early to Rab7-positive late stages. Phagosomes finally fuse with lysosomes to form phagolysosomes, which degrade apoptotic cells; however, the molecular mechanism underlying phagosome–lysosome fusion is not fully understood. Here we show that the Caenorhabditis elegans Arf-like small GTPase Arl8 (ARL-8) is involved in phagolysosome formation and is required for the efficient removal of apoptotic cells. Loss of function of arl-8 results in the accumulation of apoptotic germ cells. Both the engulfment of the apoptotic cells by surrounding somatic sheath cells and the phagosomal maturation from RAB-5- to RAB-7-positive stages occur in arl-8 mutants. However, the phagosomes fail to fuse with lysosomes in the arl-8 mutants, leading to the accumulation of RAB-7-positive phagosomes and the delayed degradation of apoptotic cells. ARL-8 localizes primarily to lysosomes and physically interacts with the homotypic fusion and protein sorting complex component VPS-41. Collectively our findings reveal that ARL-8 facilitates apoptotic cell removal in vivo by mediating phagosome–lysosome fusion during phagocytosis.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion1,2. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.

The Ras-homologous GTPase Rheb that is conserved from yeast to human appears to be involved not only in cell growth but also in nutrient uptake. Recent biochemical analysis revealed that tuberous sclerosis complex (TSC), a GTPase-activating protein (GAP), deactivates Rheb and that phosphatidylinositol 3'-kinase (PI3k)-Akt/PKB kinase pathway activates Rheb through inhibition of the GAP-mediated deactivation. Although mammalian target of rapamycin (mTOR) kinase is implicated in the downstream target of Rheb, the direct effector(s) and exact functions of Rheb have not been fully elucidated. Here we identified that Rheb expression in cultured cells induces the formation of large cytoplasmic vacuoles, which are characterized as late endocytic (late endosome- and lysosome-like) components. The vacuole formation required the GTP form of Rheb, but not the activation of the downstream mTOR kinase. These results suggest that Rheb regulates endocytic trafficking pathway independent of the previously identified mTOR pathway. The physiological roles of the two Rheb-dependent signaling pathways are discussed in terms of nutrient uptake and cell growth or cell cycle progression.

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

The forebrain, consisting of the telencephalon and diencephalon, is essential for processing sensory information. To genetically dissect formation of the forebrain in vertebrates, we carried out a systematic screen for mutations affecting morphogenesis of the forebrain in Medaka. Thirty-three mutations defining 25 genes affecting the morphological development of the forebrain were grouped into two classes. Class 1 mutants commonly showing a decrease in forebrain size, were further divided into subclasses 1A to 1D. Class 1A mutation (1 gene) caused an early defect evidenced by the lack of bf1 expression, Class 1B mutations (6 genes) patterning defects revealed by the aberrant expression of regional marker genes, Class 1C mutation (1 gene) a defect in a later stage, and Class 1D (3 genes) a midline defect analogous to the zebrafish one-eyed pinhead mutation. Class 2 mutations caused morphological abnormalities in the forebrain without considerably affecting its size, Class 2A mutations (6 genes) caused abnormalities in the development of the ventricle, Class 2B mutations (2 genes) severely affected the anterior commissure, and Class 2C (6 genes) mutations resulted in a unique forebrain morphology. Many of these mutants showed the compromised sonic hedgehog expression in the zona-limitans-intrathalamica (zli), arguing for the importance of this structure as a secondary signaling center. These mutants should provide important clues to the elucidation of the molecular mechanisms underlying forebrain development, and shed new light on phylogenically conserved and divergent functions in the developmental process.

The small GTPase Ras family regulates a variety of cell functions including proliferation and differentiation. Here we have identified novel Ras members, human Di-Ras1 and Di-Ras2, belonging to a distinct branch of the GTPase family. Di-Ras1 and Di-Ras2 specifically expressed in heart and brain share 30-40% overall identity with other members of Ras family, however, they have the following characteristic substitutions at highly conserved regions among the Ras family. 1) Thr-63 and Ser-65 in Di-Ras are substituted for Ala-59 and Gln-61 positions in Ha-Ras, respectively, that are known to be critical for GTP hydrolysis. 2) Within the effector domains, Di-Ras has Ile at a position corresponding to Asp-33 in Ha-Ras, which is important for its interaction with the downstream effector Raf. As predicted by these substitutions, Di-Ras has only a quite low level of GTPase activity and exists predominantly as a GTP-bound form upon its expression in living cells. Moreover, Di-Ras fails to interact with the Ras-binding domain of Raf, resulting in no stimulation of mitogen-activated protein kinase. Interestingly, introduction of Di-Ras into HEK293T cells induces large cellular vacuolation. These findings raise the possibility that Di-Ras might regulate cell morphogenesis in a manner distinct from other members of Ras family.

The small GTPase Rab family, which cycles between GTP-bound active and GDP-bound inactive states, plays an important role in membrane trafficking. Among them, Rab5 is involved in early endocytic pathway, and several Rab5-binding proteins have been identified as regulators or effectors to coordinate the docking and fusion processes of endocytic vesicles. We describe a novel binding protein exhibiting unique biochemical properties for Rab5. The Rab5-binding protein enhances GDP-GTP exchange reaction on Rab5 but preferentially interacts with its GTP-bound form. Gel filtration and immunoprecipitation analyses indicate that the Rab5-binding protein functions as a tetramer composed of anti-parallel linkage of two parallel dimers. These results suggest that the newly identified protein may function as an upstream activator and/or downstream effector for Rab5 in endocytic pathway. Possible roles of the quaternary structure have been discussed in terms of the Rab5-mediated signaling.

Cytoplasmic ribonucleoprotein granules, known as processing bodies (P-bodies), contain a common set of conserved RNA-processing enzymes, and mRNAs with AU-rich elements (AREs) are delivered to P-bodies for translational silencing. Although the dynamics of P-bodies is physically linked to cytoskeletal network, it is unclear how small GTPases are involved in the P-body regulation and the ARE-mRNA metabolism. We found here that glucose depletion activates RhoA GTPase and alters the P-body dynamics in HeLa cells. These glucose-depleted effects are reproduced by the overexpression of the RhoA-subfamily GTPases and conversely abolished by the inhibition of RhoA activation. Interestingly, both RhoA activation and glucose depletion inhibit the mRNA accumulation and degradation. These findings indicate that RhoA participates in the stress-induced rearrangement of P-bodies and the release of nucleated ARE-mRNAs for their stabilization.

Cilia are microtubule-based organelles that are present on the surfaces of almost all vertebrate cells. Most cilia function as sensory or molecular transport structures. Malfunctions of cilia have been implicated in several diseases of human development. The assembly of cilia is initiated by the centriole (or basal body), and several centrosomal proteins are involved in this process. The mammalian LIM protein Ajuba is a well-studied centrosomal protein that regulates cell division but its role in ciliogenesis is unknown. In this study, we isolated the medaka homolog of Ajuba and showed that Ajuba localizes to basal bodies of cilia in growth-arrested cells. Knockdown of Ajuba resulted in randomized left-right organ asymmetries and altered expression of early genes responsible for left-right body axis determination. At the cellular level, we found that Ajuba function was essential for ciliogenesis in the cells lining Kupffer's vesicle; it is these cells that induce the asymmetric fluid flow required for left-right axis determination. Taken together, our findings identify a novel role for Ajuba in the regulation of vertebrate ciliogenesis and left-right axis determination.

SummaryA genome-wide screen revealed previously unidentified components required for transport and Golgi organization (TANGO). We now provide evidence that one of these proteins, TANGO1, is an integral membrane protein localized to endoplasmic reticulum (ER) exit sites, with a luminal SH3 domain and a cytoplasmic proline-rich domain (PRD). Knockdown of TANGO1 inhibits export of bulky collagen VII from the ER. The SH3 domain of TANGO1 binds to collagen VII; the PRD binds to the COPII coat subunits, Sec23/24. In this scenario, PRD binding to Sec23/24 subunits could stall COPII carrier biogenesis to permit the luminal domain of TANGO1 to guide SH3-bound cargo into a growing carrier. All cells except those of hematopoietic origin express TANGO1. We propose that TANGO1 exports other cargoes in cells that do not secrete collagen VII. However, TANGO1 does not enter the budding carrier, which represents a unique mechanism to load cargo into COPII carriers.

The small GTPase Rab5 plays important roles in membrane budding and trafficking in the early endocytic pathways, and the activation of this GTPase is mediated by several guanine nucleotide exchange factors (GEFs) at each of the transport steps. The RIN family has been identified as GEFs for Rab5 and shown to possess unique biochemical properties. The RIN family preferentially interacts with an activated form of Rab5, although it enhances guanine nucleotide exchange reaction. Moreover, biochemical analysis indicates that the RIN family functions as a tetramer. In this chapter, we describe the isolation of the recombinant RIN family via expression in Spodoptera frugiperda (Sf9) insect cells and in mammalian cells. In addition, functional analysis is also provided to assess the physiological properties of the RIN family.

The small GTPase Rab5, which cycles between active (GTP-bound) and inactive (GDP-bound) states, plays essential roles in membrane budding and trafficking in the early endocytic pathway. However, the molecular mechanisms underlying the Rab5-regulated processes are not fully understood other than the targeting event to early endosomes. Here, we report a novel Rab5-binding protein, RIN3, that contains many functional domains shared with other RIN members and additional Pro-rich domains. RIN3 displays the same biochemical properties as RIN2, the stimulator and stabilizer of GTP-Rab5. In addition, RIN3 exhibits its unique intracellular localization. RIN3 expressed in HeLa cells localized to cytoplasmic vesicles and the RIN3-positive vesicles contained Rab5 but not the early endosomal marker EEA1. Transferrin appeared to be transported partly through the RIN3-positive vesicles to early endosomes. RIN3 was also capable of interacting via its Pro-rich domain with amphiphysin II, which contains SH3 domain and participates in receptor-mediated endocytosis. Interestingly, cytoplasmic amphiphysin II was translocated into the RIN3- and Rab5-positive vesicles when co-expressed with RIN3. These results indicate that RIN3 biochemically characterized as the stimulator and stabilizer for GTP-Rab5 plays an important role in the transport pathway from plasma membrane to early endosomes.