Several ADP-ribosylation factor (Arf) isoforms support COPI vesicle formation

Newly synthesized proteins and lipids are transported in vesicular carriers along the secretory pathway. Arfs (ADP-ribosylation factors), a family of highly conserved GTPases within the Ras superfamily, control recruitment of molecular coats to membranes, the initial step of coated vesicle biogenesis. Arf1 and coatomer constitute the minimal cytosolic machinery leading to COPI vesicle formation from Golgi membranes. Although some functional redundancies have been suggested, other Arf isoforms have been poorly analyzed in this context. In this study, we found that Arf1, Arf4, and Arf5, but not Arf3 and Arf6, associate with COPI vesicles generated in vitro from Golgi membranes and purified cytosol. Using recombinant myristoylated proteins, we show that Arf1, Arf4, and Arf5 each support COPI vesicle formation individually. Unexpectedly, we found that Arf3 could also mediate vesicle biogenesis. However, Arf3 was excluded from the vesicle fraction in the presence of the other isoforms, highlighting a functional competition between the different Arf members.     

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.     

Multiple and stepwise interactions between coatomer and ADP-ribosylation factor-1 (Arf1)-GTP

The small GTPase ADP-ribosylation factor-1 (Arf1) plays a key role in the formation of coat protein I (COP I)-coated vesicles. Upon recruitment to the donor Golgi membrane by interaction with dimeric p24 proteins, Arf1's GDP is exchanged for GTP. Arf1-GTP then dissociates from p24, and together with other Golgi membrane proteins, it recruits coatomer, the heptameric coat protein complex of COP I vesicles, from the cytosol. In this process, Arf1 was shown to specifically interact with the coatomer beta and gamma-COP subunits through its switch I region, and with epsilon-COP. Here, we mapped the interaction of the Arf1-GTP switch I region to the trunk domains of beta and gamma-COP. Site-directed photolabeling at position 167 in the C-terminal helix of Arf1 revealed a novel interaction with coatomer via a putative longin domain of delta-COP. Thus, coatomer is linked to the Golgi through multiple interfaces with membrane-bound Arf1-GTP. These interactions are located within the core, adaptor-like domain of coatomer, indicating an organizational similarity between the COP I coat and clathrin adaptor complexes.     

The ADP-ribosylation factor GTPase-activating protein Glo3p is involved in ER retrieval.

Retrograde transport of proteins from the Golgi to the endoplasmic reticulum (ER) has been the subject of some interest in the recent past. Here a new thermosensitive yeast mutant defective in retrieval of dilysine-tagged proteins from the Golgi back to the endoplasmic reticulum was characterized. The ret4-1 mutant also exhibited a selective defect in forward ER-to-Golgi transport of some secreted proteins at the non-permissive temperature. The corresponding RET4 gene was found to encode Glo3p, a GTPase-activating protein (GAP) specific for ADP-ribosylation factor (ARF). In vitro, the Glo3 thermosensitive mutant showed a reduced ARF1-GAP activity. The Glo3 protein belongs to a family of zinc finger proteins that may include additional ARF-GAPs. Gene deletion experiments of other family members showed that only GLO3 deletion resulted in impaired retrieval of dilysine-tagged proteins back to the ER. These results demonstrate that Glo3p is the main ARF-GAP specifically involved in ER retrieval.     

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [³²P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.     

The Saccharomyces cerevisiae Rheb G-protein is involved in regulating canavanine resistance and arginine uptake

The new member of the Ras superfamily of G-proteins, Rheb, has been identified in rat and human, but its function has not been defined. We report here the identification of Rheb homologues in the budding yeast Saccharomyces cerevisiae (ScRheb) as well as in Schizosaccharomyces pombe, Drosophila melanogaster, zebrafish, and Ciona intestinalis. These proteins define a new class of G-proteins based on 1) their overall sequence similarity, 2) high conservation of their effector domain sequence, 3) presence of a unique arginine in their G1 box, and 4) presence of a conserved CAAX farnesylation motif. Characterization of an S. cerevisiae strain deficient in ScRheb showed that it is hypersensitive to growth inhibitory effects of canavanine and thialysine, which are analogues of arginine and lysine, respectively. Accordingly, the uptake of arginine and lysine was increased in the ScRheb-deficient strain. This increased arginine uptake requires the arginine-specific permease Can1p. The function of ScRheb is dependent on having an intact effector domain since mutations in the effector domain of ScRheb are incapable of complementing canavanine hypersensitivity of scrheb disruptant cells. Furthermore, the conserved arginine in the G1 box plays a role in the activity of ScRheb, as a mutation of this arginine to glycine significantly reduced the ability of ScRheb to complement canavanine hypersensitivity of ScRheb-deficient yeast. Finally, a mutation in the C-terminal CAAX farnesylation motif resulted in a loss of ScRheb function. This result, in combination with our finding that ScRheb is farnesylated, suggests that farnesylation plays a key role in ScRheb function. Our findings assign the regulation of arginine and lysine uptake as the first physiological function for this new farnesylated Ras superfamily G-protein.     

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.     

Factors regulating copper uptake in a cyanobacterium

Copper uptake in the diazotrophic cyanobacteriumNostoc calcicola was found to be typically biphasic, comprising rapid binding of the cations to the cell wall (during the first 10 min) followed by the subsequent metabolism-dependent intracellular uptake for at least 1 h, with a curvilinear kinetics saturating at 40 µM (K_m 25.0 µM, V_max 3.0 nmol Cu mg^–1 protein min^–1). The cellular Cu uptake was light- and ATP-dependent, and the addition of 3(3,4-dichlorophenyl)-1,1-dimethylurea or exogenous ATP proved that the energy to drive Cu transport was derived mainly through PS II reactions. The application of metabolic inhibitors and uncouplers like carbonylcyanidep-nitrofluoromethoxylphenyl hydrazone, N,N-dicyclohexycarbodiimide, azide, and p-chloromercuribenzoate revealed that -SH group(s), proton gradient across the cell membrane, and ATP hydrolysis were involved in the transmembrane movement of Cu inN. calcicola. While monothiol (2-mercaptoethanol) caused a twofold reduction in Cu uptake rate, dithiol (dithiothreitol) contributed towards a further drop in the cation uptake rate.     

bZIP转录因子CfHac1参与调控果生刺盘孢菌的生长发育和致病力

油茶炭疽病是油茶Camellia oleifera上最重要病害之一,引起该病害的主要致病菌为果生刺盘孢菌Colletotrichum fructicola。本研究以果生刺盘孢菌bZIP类转录因子CfHac1为研究对象,研究其在果生刺盘孢菌的营养生长、产孢量、附着胞形成、致病力及耐受性等方面的生物学功能,为油茶炭疽病的防控提供理论依据。研究结果表明,果生刺盘孢菌中具有一个与灰色大角间座壳（稻瘟菌）bZIP转录因子MoHac1直系同源的基因,命名为CfHAC1。该基因全长1 627bp,编码526个氨基酸,该蛋白含有一个碱性亮氨酸链（bZIP）结构域和3个未知功能结构域。CfHAC1基因敲除突变体的菌丝生长速度显著变慢,分生孢子产量显著减少且不能正常形成附着胞,并对山梨糖醇和KCl渗透压胁迫敏感性增加;致病力测试结果表明,果生刺盘孢菌基因敲除突变体ΔCfhac1对油茶的致病力显著下降。转录因子CfHac1参与调控果生刺盘孢菌的生长、产孢、附着胞的形成、致病力以及响应外界渗透压胁迫过程。     

Primary platelet signaling cascades and integrin-mediated signaling control ADP-ribosylation factor (Arf) 6-GTP levels during platelet activation and aggregation

Previous studies showed that ADP-ribosylation factor 6 (Arf6) is important for platelet function; however, little is known about which signaling events regulate this small GTP-binding protein. Arf6-GTP was monitored in platelets stimulated with a number of agonists (TRAP, thrombin, convulxin, collagen, PMA, thapsigargin, or A23187) and all led to a time-dependent decrease in Arf6-GTP. ADP and U46619 were without effect. Using inhibitors, it was shown that the decrease of Arf6-GTP is a direct consequence of known signaling cascades. Upon stimulation via PAR receptors, Arf6-GTP loss could be blocked by treatment with U-73122, BAPTA/AM, Ro-31-8220, or Gö6976, indicating requirements for phospholipase C, calcium, and protein kinase C (PKC) α/β, respectively. The Arf6-GTP decrease in convulxin-stimulated platelets showed similar requirements and was also sensitive to piceatannol, wortmannin, and {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, indicating additional requirements for Syk and phosphatidylinositol 3-kinase. The convulxin-induced decrease was sensitive to both PKCα/β and δ inhibitors. Outside-in signaling, potentially via integrin engagement, caused a second wave of signaling that affected Arf6. Inclusion of RGDS peptides or EGTA, during activation, led to a biphasic response; Arf6-GTP levels partially recovered upon continued incubation. A similar response was seen in β3 integrin-null platelets. These data show that Arf6-GTP decreases in response to known signaling pathways associated with PAR and GPVI. They further reveal a second, aggregation-dependent, process that dampens Arf6-GTP recovery. This study demonstrates that the nucleotide state of Arf6 in platelets is regulated during the initial phases of activation and during the later stages of aggregation.Platelet activation is initiated through several classes of membrane receptors, which are stimulated by agonists produced at the vascular lesion (1–3). A second wave of signaling, caused by engagement of integrins, occurs as platelets bind to the lesion surface and aggregate (4). Together, these plasma membrane proteins initiate the platelet processes important for thrombosis (e.g. adhesion, spreading, secretion, and clot retraction). Small GTP-binding proteins, specifically members of the Ras superfamily, link signaling events from various platelet receptors to defined outcomes, such as shape change (5–7), aggregation (8, 9), and secretion (10–12). Rab proteins play roles in granule secretion, with Rab4 and Rab6 being involved in alpha granule release (10, 11) and Rab27a/b in dense core granule release (12, 13). RalA is activated in response to various stimuli (14–16) and may play a role in secretion by anchoring the exocyst complex to specific membrane sites (17). Rap1 plays a role in integrin α_IIbβ₃ activation (8, 9). Rho family GTPases (Rho, Rac, and Cdc42) play roles in platelet phosphoinositide signaling and in the regulation of the actin cytoskeleton (5–7). While these small GTP-binding proteins are clearly important to platelet function, it is equally clear that other small G proteins are present and functional in platelets (18).The ADP-ribosylation factor (Arf)² family are Ras-related, small GTPases that affect both vesicular transport and cytoskeletal dynamics (19, 20). Based on their primary sequences, this family is divided into three classes, with Arf6 as the only member of class III (19). Arf6-GTP is considered the “active state” and can interact with downstream effectors, such as phospholipase D (PLD) (21), phosphatidylinositol 4-phosphate 5-kinase type α (22), and arfaptin 2 (23, 24), resulting in the recruitment of these effectors to the plasma membrane. The Arf6 GTP/GDP cycle is mediated by interactions with guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). The large number of Arf-GEF and -GAP proteins have been discussed in recent reviews where it was noted that, unlike other small GTPases, Arf functions are generally not mediated solely by the GTP-bound state but through its cycling between states (19, 20, 25, 26).The effects that Arf6 has on the secretion and actin dynamics in nucleated cells make it an ideal candidate for function in platelets. Arf6 influences cortical actin and is important for spreading, ruffling, migration, and phagocytosis (reviewed in Ref. 19). Our previous work (27) showed that Arf6 is present on platelet membranes and is important for platelet function. Unlike other small G proteins, the Arf6 GTP-bound form is readily detectible in resting platelets and upon activation with collagen or convulxin there is a rapid conversion to the GDP-bound form. Acylated peptides, which mimic the myristoylated N terminus of Arfs have been used as isoform-specific inhibitors (28). In platelets, a myristoylated-Arf6 (myr-Arf6) peptide specifically blocks the activation-dependent loss of Arf6-GTP. This peptide also blocks aggregation, spreading on collagen, and activation of the Rho family of GTPases. Other GTPases, such as Ral and Rap, were unaffected. The simplest explanation for these data is that platelet activation stimulates the GTPase activity of Arf6, perhaps through activation of an Arf6-GAP. Alternatively, platelet activation could affect an Arf6-GEF thus reducing the production of Arf6-GTP. Regardless of mechanism, disruption of the activation-dependent loss of Arf6-GTP, with the myr-Arf6 peptide, profoundly affects the actin-based cytoskeletal rearrangements associated with platelet activation. While our initial report (27) established a role for Arf6 in platelet function, it was not clear what platelet signaling events were required to induce the loss of Arf6-GTP.In this article, we delineate the signaling cascades required for the activation-dependent loss of Arf6-GTP. We show that the Arf6-GTP to -GDP conversion was stimulated by primary agonists (thrombin, TRAP, collagen, or convulxin) but not by ADP or U46619. The decrease in Arf6-GTP, downstream of thrombin and convulxin, required PLC, and PKC activity. Loss of Arf6-GTP, via stimulation of GPVI with convulxin, additionally required Syk and PI3K activities. Pretreatment with passivators, nitric oxide (NO), and prostaglandin I₂ (PGI₂) blocked thrombin- and convulxin-induced loss of Arf6-GTP. Further experiments suggested a role for “outside-in” signaling, especially once platelet aggregates begin to form. Inclusion of RGDS peptide, EGTA, or the deletion of the β3 integrin had only minimal effects on the initial loss of Arf6-GTP but led to the partial recovery of Arf6-GTP levels. This biphasic change in Arf6-GTP levels was not seen when aggregation was allowed to occur normally. Taken together, these data show that the Arf6 nucleotide state is responsive to both initial agonist-mediated signaling and to a second wave of integrin-mediated signaling that occurs upon aggregation.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

The BRCT domain of mammalian Rev1 is involved in regulating DNA translesion synthesis

Rev1 is a deoxycytidyl transferase associated with DNA translesion synthesis (TLS). In addition to its catalytic domain, Rev1 possesses a so-called BRCA1 C-terminal (BRCT) domain. Here, we describe cells and mice containing a targeted deletion of this domain. Rev1^B/B mice are healthy, fertile and display normal somatic hypermutation. Rev1^B/B cells display an elevated spontaneous frequency of intragenic deletions at Hprt. In addition, these cells were sensitized to exogenous DNA damages. Ultraviolet-C (UV-C) light induced a delayed progression through late S and G2 phases of the cell cycle and many chromatid aberrations, specifically in a subset of mutant cells, but not enhanced sister chromatid exchanges (SCE). UV-C-induced mutagenesis was reduced and mutations at thymidine–thymidine dimers were absent in Rev1^B/B cells, the opposite phenotype of UV-C-exposed cells from XP-V patients, lacking TLS polymerase η. This suggests that the enhanced UV-induced mutagenesis in XP-V patients may depend on error-prone Rev1-dependent TLS. Together, these data indicate a regulatory role of the Rev1 BRCT domain in TLS of a limited spectrum of endogenous and exogenous nucleotide damages during a defined phase of the cell cycle.     

Factors regulating copper uptake in free and immobilized cyanobacterium

The effect of population size, redox potential, exogenous ATP and complexing agents on Cu uptake by free and immobilized cyanobacteriumNostoc calcicola Bréb. has been studied. Cu uptake was regulated by the population size. In such comparisons, the immobilized cells had a greater longevity. Low pH conditions enhanced Cu uptake. Exogenous ATP (10 μmol/L) supplied to dark-grown free and immobilized cells did not support Cu uptake to the extent of light-grown cells. Experiments involving natural as well as synthetic complexing agents clearly established the superiority of soil extract and spent medium over EDTA (10 μmol/L), in sequestering Cu in free as well as immobilized cells.     

The factor IX gamma-carboxyglutamic acid (Gla) domain is involved in interactions between factor IX and factor XIa

During hemostasis, factor IX is activated to factor IXabeta by factor VIIa and factor XIa. The glutamic acid-rich gamma-carboxyglutamic acid (Gla) domain of factor IX is involved in phospholipid binding and is required for activation by factor VIIa. In contrast, activation by factor XIa is not phospholipid-dependent, raising questions about the importance of the Gla for this reaction. We examined binding of factors IX and IXabeta to factor XIa by surface plasmon resonance. Plasma factors IX and IXabeta bind to factor XIa with K(d) values of 120 +/- 11 nm and 110 +/- 8 nm, respectively. Recombinant factor IX bound to factor XIa with a K(d) of 107 nm, whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and factor IX expressed in the presence of warfarin (rFIX-desgamma) did not bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor of factor IX binding to factor XIa (K(i) 34 nm) and activation by factor XIa (K(i) 33 nm). In activated partial thromboplastin time clotting assays, the specific activities of plasma and recombinant factor IX were comparable (200 and 150 units/mg), whereas rFIX/VII-Gla activity was low (<2 units/mg). In contrast, recombinant factor IXabeta and activated rFIX/VIIa-Gla had similar activities (80 and 60% of plasma factor IXabeta), indicating that both proteases activate factor X and that the poor activity of zymogen rFIX/VII-Gla was caused by a specific defect in activation by factor XIa. The data demonstrate that factor XIa binds with comparable affinity to factors IX and IXabeta and that the interactions are dependent on the factor IX Gla domain.