堅田 利明

Mammalian endoplasmic reticulum (ER) exit sites export a variety of cargo molecules including oversized cargoes such as collagens. However, the mechanisms of their assembly and organization are not fully understood. TAN GO1L is characterized as a collagen receptor, but the function of TAN GO1S remains to be investigated. Here, we show that direct interaction between both isoforms of TAN GO1 and Sec16 is not only important for their correct localization but also critical for the organization of ER exit sites. The depletion of TAN GO1 disassembles COPII components as well as membrane-bound ER-resident complexes, resulting in fewer functional ER exit sites and delayed secretion. The ectopically expressed TAN GO1 C-terminal domain responsible for Sec16 binding in mitochondria is capable of recruiting Sec16 and other COPII components. Moreover, TAN GO1 recruits membrane-bound macromolecular complexes consisting of cTAGE5 and Sec12 to the ER exit sites. These data suggest that mammalian ER exit sites are organized by TAN GO1 acting as a scaffold, in cooperation with Sec16 for efficient secretion.

Collagens synthesized within the endoplasmic reticulum (ER) are too large to fit in conventional COPII-coated transport vesicles; thus their export from the ER requires specialized factors. TANGO1 (L) is an integral membrane protein that binds to collagen and the coatomer of vesicles and is necessary for collagen secretion from the ER. Here we characterized the short isoform of TANGO1 (TANGO1S), lacking the collagen-binding domain, and found that it was independently required for collagen export from the ER. Moreover, we found that each of the TANGO1 isoforms forms a stable protein complex with factors involved in collagen secretion: TANGO1L/cTAGE5/Sec12 (900 kDa) and TANGO1S/cTAGE5/Sec12 (700 kDa). Of interest, TANGO1S and TANGO1L seemed to be interchangeable in exporting collagen from the ER. Our results suggest that mammalian ER exit sites possess two different-sized membrane-bound macromolecular complexes that specifically function in large-cargo export from the ER.

Two independent functions of cTAGE5 have been reported in collagen VII export from the endoplasmic reticulum (ER). cTAGE5 not only forms a cargo receptor complex with TANGO1, but it also acts as a scaffold to recruit Sec12, a guanine-nucleotide exchange factor for Sar1 GTPase, to ER exit sites. However, the relationship between the two functions remains unclear. Here we isolated point mutants of cTAGE5 that lost Sec12-binding ability but retained binding to TANGO1. Although expression of the mutant alone could not rescue the defects in collagen VII secretion mediated by cTAGE5 knockdown, coexpression with Sar1, but not with the GTPase-deficient mutant, recovered secretion. The expression of Sar1 alone failed to rescue collagen secretion in cTAGE5-depleted cells. Taken together, these results suggest that two functionally irreplaceable and molecularly separable modules in cTAGE5 are both required for collagen VII export from the ER. The recruitment of Sec12 by cTAGE5 contributes to efficient activation of Sar1 in the vicinity of ER exit sites. In addition, the GTPase cycle of Sar1 appears to be responsible for collagen VII exit from the ER.

Adult T-cell leukemia/lymphoma (ATLL) is a distinct form of peripheral T-cell lymphoma with poor prognosis, which is caused by the human T-lymphotropic virus type 1 (HTLV-1). In contrast to the unequivocal importance of HTLV-1 infection in the pathogenesis of ATLL, the role of acquired mutations in HTLV-1 infected T cells has not been fully elucidated, with a handful of genes known to be recurrently mutated. In this study, we identified unique RHOA mutations in ATLL through whole genome sequencing of an index case, followed by deep sequencing of 203 ATLL samples. RHOA mutations showed distinct distribution and function from those found in other cancers. Involving 15% (30/203) of ATLL cases, RHOA mutations were widely distributed across the entire coding sequence but almost invariably located at the guanosine triphosphate (GTP)-binding pocket, with Cys16Arg being most frequently observed. Unexpectedly, depending on mutation types and positions, these RHOA mutants showed different or even opposite functional consequences in terms of GTP/guanosine diphosphate (GDP)-binding kinetics, regulation of actin fibers, and transcriptional activation. The Gly17Val mutant did not bind GTP/GDP and act as a dominant negative molecule, whereas other mutants (Cys16Arg and Ala161Pro) showed fast GTP/GDP cycling with enhanced transcriptional activation. These findings suggest that both loss-and gain-of-RHOA functions could be involved in ATLL leukemogenesis. In summary, our study not only provides a novel insight into the molecular pathogenesis of ATLL but also highlights a unique role of variegation of heterologous RHOA mutations in human cancers.

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.

The Ras family of small GTPases function in a wide variety of biological processes as "molecular switches" by cycling between inactive GDP-bound and active GTP-bound forms. Di-Ras1 and Di-Ras2 were originally identified as small GTPases forming a distinct subgroup of the Ras family. Di-Ras1/Di-Ras2 mRNAs are detected predominantly in brain and heart tissues. Biochemical analysis of Di-Ras1/Di-Ras2 has revealed that they have little GTPase activity and that their intrinsic guanine-nucleotide exchange rates are much faster than that of H-Ras. Yet little is known about the biological role(s) of Di-Ras1/Di-Ras2 or of how their activities are regulated. In the present study we found that endogenous Di-Ras2 co-purifies with SmgGDS from rat brain cytosol. Size-exclusion chromatography of purified recombinant proteins showed that Di-Ras2 forms a high affinity complex with SmgGDS. SmgGDS is a guanine nucleotide exchange factor with multiple armadillo repeats and has recently been shown to specifically activate RhoA and RhoC. In contrast to the effect on RhoA, SmgGDS does not act as a guanine nucleotide exchange factor for Di-Ras2 but instead tightly associates with Di-Ras2 to reduce its binding affinity for guanine nucleotides. Finally, pulse-chase analysis revealed that Di-Ras2 binds, in a C-terminal CAAX motif-dependent manner, to SmgGDS immediately after its synthesis. This leads to increased Di-Ras2 stability. We thus propose that isoprenylated Di-Ras2 forms a tight complex with SmgGDS in cytosol immediately after its synthesis, which lowers its affinity for guanine nucleotides.

The nutritional status of an organism can greatly impact the function and behavior of stem and progenitor cells [1]. However, the regulatory circuits that inform these cells about the dietary environment remain to be elucidated. Newly hatched C. elegans larvae (L1s) halt development in "L1 arrest'' or "L1 diapause'' until ample food is encountered and triggers stem and progenitor cells to exit from quiescence [2]. The insulin/insulin-like growth factor signaling (IIS) pathway plays a key role in this reactivation [3, 4], but its site(s) of action have not been elucidated nor have the nutrient molecule(s) that stimulate the pathway been identified. By tissue-specifically modulating the activity of its components, we demonstrate that the IIS pathway acts in the hypodermis to regulate nutrition-responsive reactivation of neural and mesodermal progenitor cells. We identify ethanol, a likely component of the natural Caenorhabditis habitat, and amino acids as nutrients that synergistically reactivate somatic progenitor cells and upregulate expression of insulin-like genes in starved L1 larvae. The hypodermis likely senses the availability of amino acids because forced activation of the amino-acid-responsive Rag-TORC1 (target of rapamycin complex 1) pathway in this tissue can also release somatic progenitor cell quiescence in the presence of ethanol. Finally, there appears to be crosstalk between the IIS and Rag-TORC1 pathways because constitutive activation of the IIS pathway requires Rag to promote reactivation. This work demonstrates that ethanol and amino acids act as dietary cues via the IIS and Rag-TORC1 pathways in the hypodermis to coordinately control progenitor cell behavior.

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.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms. © 2013 Cevik et al.

The alpha-subunit of G(i-2), in addition to that of G(s) (GTP-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in HL-60 cell membranes when a chemotactic receptor was stimulated by formyl-Met-Leu-Phe (fMLP), and the sites modified by cholera and pertussis toxins on the alpha-subunit of G(i-2) were different (Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 21394-21400). In order to investigate how the functions of G(i-2) were modified by cholera toxin, the ADP-ribosylated and unmodified proteins were purified from HL-60 cell membranes that had been incubated in the presence and absence of cholera toxin, respectively. The modified G(i-2) displayed unique properties as follows. 1) The ADP-ribosylated alpha-subunit had a more acidic pI than the unmodified one, leading to a partial resolution of the modified G(i-2) trimer from the unmodified protein by an anion column chromatography. 2) When the purified proteins were incubated with [gamma-P-32] GTP, the radioactivity was more greatly retained in the modified G(i-2) than in the unmodified protein. 3) The actual catalytic rate (k(cat) of GTP hydrolysis was, indeed, markedly inhibited by cholera toxin-induced modification. 4) There was an increase in the apparent affinity of G(i-2) for GDP by cholera toxin-induced modification. 5) The modified G(i-2) exhibited a low substrate activity for pertussis toxin-catalyzed ADP-ribosylation. 6) A high-affinity fMLP binding to HL-60 cell membranes was more effectively reconstituted with the ADP-ribosylated G(i-2) than with the unmodified protein. These results suggested that the agonist-fMLP receptor complex was effectively coupled with the ADP-ribosylated G(i-2), resulting in the GTP-bound form, and that the hydrolysis of GTP on the modified alpha-subunit was selectively attenuated. Thus, cholera toxin ADP-ribosylated G(i-2) appeared to be not only a less sensitive pertussis toxin substrate but also an efficient signal transducer between receptors and effectors.

The alpha-subunits of G(i) (G(i)alpha) and G(s) (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of G(i)alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [liri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (I 989) J. Biol. Chem. 264, 21 394 - 21400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same G(i)alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) G(i)alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified G(i) was much lower than that of intact G(i). These results suggested that G(i) ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha-subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of G(i) by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of G(i) with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (G(K))-gated muscarinic K+ channel (I(K.ACh)), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) gradually increased the I(K.ACh) current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of I(K.ACh) in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma-S. The agonist-independent, GTP-induced activation of I(K.ACh) was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of I(K.ACh) was increased and the GTP concentration for the half-maximal activation of I(K.ACh) was decreased. Intracellular GDP inhibited the GTP-induced openings of I(K.ACh) in a concentration-dependent fashion. The half-inhibition of I(K.ACh) openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and G(K) is essential for basal stimulation of I(K.ACh), and (b) that ACh binding to the receptor accelerates the turnover of G(K) and increases G(K)'s affinity to GTP analogues over GDP.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with M(r) values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.