Reconstitution of human atlastin fusion activity reveals autoinhibition by the C terminus

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.     

The dynamin-like GTPase Sey1p mediates homotypic ER fusion in S. cerevisiae

The endoplasmic reticulum (ER) forms a network of tubules and sheets that requires homotypic membrane fusion to be maintained. In metazoans, this process is mediated by dynamin-like guanosine triphosphatases (GTPases) called atlastins (ATLs), which are also required to maintain ER morphology. Previous work suggested that the dynamin-like GTPase Sey1p was needed to maintain ER morphology in Saccharomyces cerevisiae. In this paper, we demonstrate that Sey1p, like ATLs, mediates homotypic ER fusion. The absence of Sey1p resulted in the ER undergoing delayed fusion in vivo and proteoliposomes containing purified Sey1p fused in a GTP-dependent manner in vitro. Sey1p could be partially replaced by ATL1 in vivo. Like ATL1, Sey1p underwent GTP-dependent dimerization. We found that the residual ER-ER fusion that occurred in cells lacking Sey1p required the ER SNARE Ufe1p. Collectively, our results show that Sey1p and its homologues function analogously to ATLs in mediating ER fusion. They also indicate that S. cerevisiae has an alternative fusion mechanism that requires ER SNAREs.     

The hypervariable region of atlastin-1 is a site for intrinsic and extrinsic regulation

Atlastin (ATL) GTPases catalyze homotypic membrane fusion of the peripheral endoplasmic reticulum (ER). GTP-hydrolysis–driven conformational changes and membrane tethering are prerequisites for proper membrane fusion. However, the molecular basis for regulation of these processes is poorly understood. Here we establish intrinsic and extrinsic modes of ATL1 regulation that involve the N-terminal hypervariable region (HVR) of ATLs. Crystal structures of ATL1 and ATL3 exhibit the HVR as a distinct, isoform-specific structural feature. Characterizing the functional role of ATL1’s HVR uncovered its positive effect on membrane tethering and on ATL1’s cellular function. The HVR is post-translationally regulated through phosphorylation-dependent modification. A kinase screen identified candidates that modify the HVR site specifically, corresponding to the modifications on ATL1 detected in cells. This work reveals how the HVR contributes to efficient and potentially regulated activity of ATLs, laying the foundation for the identification of cellular effectors of ATL-mediated membrane processes.     

Structures of the yeast dynamin-like GTPase Sey1p provide insight into homotypic ER fusion

Homotypic membrane fusion of the endoplasmic reticulum is mediated by dynamin-like guanosine triphosphatases (GTPases), which include atlastin (ATL) in metazoans and Sey1p in yeast. In this paper, we determined the crystal structures of the cytosolic domain of Sey1p derived from Candida albicans. The structures reveal a stalk-like, helical bundle domain following the GTPase, which represents a previously unidentified configuration of the dynamin superfamily. This domain is significantly longer than that of ATL and critical for fusion. Sey1p forms a side-by-side dimer in complex with GMP-PNP or GDP/AlF₄⁻ but is monomeric with GDP. Surprisingly, Sey1p could mediate fusion without GTP hydrolysis, even though fusion was much more efficient with GTP. Sey1p was able to replace ATL in mammalian cells, and the punctate localization of Sey1p was dependent on its GTPase activity. Despite the common function of fusogenic GTPases, our results reveal unique features of Sey1p.     

Reciprocal regulation between lunapark and atlastin facilitates ER three-way junction formation

Three-way junctions are characteristic structures of the tubular endoplasmic reticulum (ER) network. Junctions are formed through atlastin (ATL)-mediated membrane fusion and stabilized by lunapark (Lnp). However, how Lnp is preferentially enriched at three-way junctions remains elusive. Here, we showed that Lnp loses its junction localization when ATLs are deleted. Reintroduction of ATL1 R77A and ATL3, which have been shown to cluster at the junctions, but not wild-type ATL1, relocates Lnp to the junctions. Mutations in the Nmyristoylation site or hydrophobic residues in the coiled coil (CC1) of Lnp N-terminus (NT) cause mis-targeting of Lnp. Conversely, deletion of the lunapark motif in the C-terminal zinc finger domain, which affects the homooligomerization of Lnp, does not alter its localization. Purified Lnp-NT attaches to the membrane in a myristoylation-dependent manner. The mutation of hydrophobic residues in CC1 does not affect membrane association, but compromises ATL interactions. In addition, Lnp-NT inhibits ATL-mediated vesicle fusion in vitro. These results suggest that CC1 in Lnp-NT contacts junction-enriched ATLs for proper localization; subsequently, further ATL activity is limited by Lnp after the junction is formed. The proposed mechanism ensures coordinated actions of ATL and Lnp in generating and maintaining three-way junctions.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins, belonging to the class-1 (eRF1) and class-2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to GDP and inorganic phosphate in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA and peptidyl-tRNA but needs the presence of eRF1. It's known that eRF1 and eRF3 interact with each other in vitro and in vivo via their C-terminal regions. eRF1 consists of three domains - N, M, and C. In this study we examined the influence of individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N-, M-, C- and NM-domains induces eRF3 GTPase activity in presence of the ribosomes. MC-domain does induce GTPase activity of eRF3 but four times less efficient than full-length eRF1, therefore, MC-domain (and very likely M-domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated, postulating that GTPase activity eRF3 during the translation termination is associated with the intermolecular interactions of GTP/GDP, GTPase center of the large ribosomal subunit (60S), MC-domain of eRF1, C-terminal region and GTP-binding domains of eRF3, but without participation of the N-terminal region of eRF3.     

Influence of individual domains of the translation termination factor eRF1 on induction of the GTPase activity of the translation termination factor eRF3

Translation termination in eukaryotes is governed by two proteins belonging to class 1 (eRF1) and class 2 (eRF3) polypeptide release factors. eRF3 catalyzes hydrolysis of GTP to yield GDP and P_i in the ribosome in the absence of mRNA, tRNA, aminoacyl-tRNA, and peptidyl-tRNA and requires eRF1 for this activity. It is known that eRF1 and eRF3 interact with each other via their C-terminal regions both in vitro and in vivo. eRF1 consists of three domains—N, M, and C. In this study we examined the influence of the individual domains of the human eRF1 on induction of the human eRF3 GTPase activity in the ribosome in vitro. It was shown that none of the N, M, C, and NM domains induces the eRF3 GTPase activity in the presence of ribosomes. The MC domain does induce the eRF3 GTPase activity, but four times less efficiently than full-length eRF1. Therefore, we assumed that the MC domain (and very likely the M domain) binds to the ribosome in the presence of eRF3. Based on these data and taking into account the data available in the literature, a conclusion was drawn that the N domain of eRF1 is not essential for eRF1-dependent induction of the eRF3 GTPase activity. A working hypothesis is formulated that the eRF3 GTPase activity in the ribosome during translation termination is associated with the intermolecular interactions of GTP/GDP, the GTPase center of the large (60S) subunit, the MC domain of eRF1, and the C-terminal region and GTP-binding motifs of eRF3 but without participation of the N-terminal region of eRF1.     

Measuring Ras-family GTP levels in vivo--running hot and cold

The detection of Ras-family GTPase activity is important in the determination of cell signaling events elicited by numerous ligands and cellular processes. This has been made much easier in recent years by the use of glutathione S-transferase (GST)-fused Ras binding domains. These domains from downstream effectors such as Raf and RalGDS preferentially bind the GTP-bound Ras proteins enabling their extraction and subsequent quantification by immunoblotting. Despite this advance, effectors that efficiently discriminate between GTP- and GDP-bound states are not available for many Ras-family members. While this hampers the ability to detect activity in tissue specimens, it is still possible to metabolically label cells with (32)Pi to load the GTP/GDP pool with labeled nucleotides, immunoprecipitate the Ras protein and detect the bound label following thin layer chromatographic separation and exposure to film or a phosphorimager. Using a transfection system and antibodies that recognize epitope tags one can test the ability of a protein to work as a GEF or GAP for a certain GTPase. Alternatively, if an immunoprecipitating antibody is available to the target GTPase, then analysis of endogenous GTP/GDP ratio is possible. Here we describe the detection of M-Ras and Rap1 activity by GST-RBD pull-down as well as that of Rheb and epitope-tagged R-Ras by classical metabolic labeling and immunoprecipitation.     

Activation of the superoxide-generating NADPH oxidase by chimeric proteins consisting of segments of the cytosolic component p67(phox) and the small GTPase Rac1.

Activation of the superoxide (O2(-))-generating NADPH oxidase of phagocytes is the consequence of the assembly of a membrane-associated flavocytochrome b(559) with the cytosolic proteins p47(phox) and p67(phox) and the small GTPase Rac (1 or 2). We proposed that Rac1 serves as a membrane-targeting molecule for p67(phox). This hypothesis was tested by constructing recombinant chimeric proteins, joining various functional domains of p67(phox) and Rac1, and expressing these in Escherichia coli. Chimeras were assayed for the ability to support O2(-) production by phagocyte membranes in an amphiphile-activated cell-free system in the presence or absence of p47(phox). A chimera consisting of p67(phox) truncated at residue 212 and fused to a full-length Rac1 [p67(phox)(1-212)-Rac1(1-192)] was a potent NADPH oxidase activator. A p67(phox)(1-212)-Rac1(178-192) chimera, to which Rac1 contributed only the C-terminal polybasic domain, was a weaker but consistent activator. Chimeras comprising the full length of Rac1 bound GTP/GDP, like bona fide GTPases. The activity of p67(phox)-Rac1 chimeras was dependent on the presence of the tetratricopeptide repeat and activation domains, in the p67(phox) segment, and on an intact polybasic region, at the C terminus of the Rac1 segment, but not on the insert region of Rac1. Partial activation by chimeras, in the GTP-bound form, was also possible in the absence of p47(phox). Evidence is offered in support of the proposal that the GTP- and GDP-bound forms of chimera p67(phox)(1-212)-Rac1(1-192) have distinct conformations, corresponding to the presence and absence of intrachimeric bonds, respectively.     

An intramolecular salt bridge drives the soluble domain of GTP-bound atlastin into the postfusion conformation

Endoplasmic reticulum (ER) network branching requires homotypic tethering and fusion of tubules mediated by the atlastin (ATL) guanosine triphosphatase (GTPase). Recent structural studies on the ATL soluble domain reveal two dimeric conformers proposed to correspond to a tethered prefusion state and a postfusion state. How the prefusion conformer transitions to the postfusion conformer is unknown. In this paper, we identify an intramolecular salt bridge mediated by two residues outside the GTPase domain near the point of rotation that converts the prefusion dimer to the postfusion state. Charge reversal of either residue blocked ER network branching, whereas a compensatory charge reversal to reestablish electrostatic attraction restored function. In vitro assays using the soluble domain revealed that the salt bridge was dispensable for GTP binding and hydrolysis but was required for forming the postfusion dimer. Unexpectedly, the postfusion conformation of the soluble domain was achieved when bound to the nonhydrolyzable GTP analogue guanosine 5'-[β,γ-imido]triphosphate, suggesting that nucleotide hydrolysis might not be required for the prefusion to postfusion conformational change.     

Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion

The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle-Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)-this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.     

Role of Individual Domains and Identification of Internal Gap in Human Guanylate Binding Protein-1

Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif (¹⁰³DXEKGD¹⁰⁸), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α-³²P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the ¹⁰³DXEKGD¹⁰⁸ motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer.     

Insight into Temperature Dependence of GTPase Activity in Human Guanylate Binding Protein-1

Interferon-γ induced human guanylate binding protein-1(hGBP1) belongs to a family of dynamin related large GTPases. Unlike all other GTPases, hGBP1 hydrolyzes GTP to a mixture of GDP and GMP with GMP being the major product at 37°C but GDP became significant when the hydrolysis reaction was carried out at 15°C. The hydrolysis reaction in hGBP1 is believed to involve with a number of catalytic steps. To investigate the effect of temperature in the product formation and on the different catalytic complexes of hGBP1, we carried out temperature dependent GTPase assays, mutational analysis, chemical and thermal denaturation studies. The Arrhenius plot for both GDP and GMP interestingly showed nonlinear behaviour, suggesting that the product formation from the GTP-bound enzyme complex is associated with at least more than one step. The negative activation energy for GDP formation and GTPase assay with external GDP together indicate that GDP formation occurs through the reversible dissociation of GDP-bound enzyme dimer to monomer, which further reversibly dissociates to give the product. Denaturation studies of different catalytic complexes show that unlike other complexes the free energy of GDP-bound hGBP1 decreases significantly at lower temperature. GDP formation is found to be dependent on the free energy of the GDP-bound enzyme complex. The decrease in the free energy of this complex at low temperature compared to at high is the reason for higher GDP formation at low temperature. Thermal denaturation studies also suggest that the difference in the free energy of the GTP-bound enzyme dimer compared to its monomer plays a crucial role in the product formation; higher stability favours GMP but lower favours GDP. Thus, this study provides the first thermodynamic insight into the effect of temperature in the product formation of hGBP1.     

Tripartite chimeras comprising functional domains derived from the cytosolic NADPH oxidase components p47phox, p67phox, and Rac1 elicit activator-independent superoxide production by phagocyte membranes: an essential role for anionic membrane phospholipids

The superoxide-generating NADPH oxidase is converted to an active state by the assembly of a membrane-localized cytochrome b(559) with three cytosolic components: p47(phox), p67(phox), and GTPase Rac1 or Rac2. Assembly involves two sets of protein-protein interactions: among cytosolic components and among cytosolic components and cytochrome b(559) within its lipid habitat. We circumvented the need for interactions among cytosolic components by constructing a recombinant tripartite chimera (trimera) consisting of the Phox homology (PX) and Src homology 3 (SH3) domains of p47(phox), the tetratricopeptide repeat and activation domains of p67(phox), and full-length Rac1. Upon addition to phagocyte membrane, the trimera was capable of oxidase activation in vitro in the presence of an anionic amphiphile. The trimera had a higher affinity (lower EC(50)) for and formed a more stable complex (longer half-life) with cytochrome b(559) compared with the combined individual components, full-length or truncated. Supplementation of membrane with anionic but not neutral phospholipids made activation by the trimera amphiphile-independent. Mutagenesis, truncations, and domain replacements revealed that oxidase activation by the trimera was dependent on the following interactions: 1) interaction with anionic membrane phospholipids via the poly-basic stretch at the C terminus of the Rac1 segment; 2) interaction with p22(phox) via Trp(193) in the N-terminal SH3 domain of the p47(phox) segment, supplementing the electrostatic attraction; and 3) an intrachimeric bond among the p67(phox) and Rac1 segments complementary to their physical fusion. The PX domain of the p47(phox) segment and the insert domain of the Rac1 segment made only minor contributions to oxidase assembly.     

Purification and characterization from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to rhoB p20, a ras p21-like GTP-binding protein

A novel regulatory protein for the rho proteins (rhoA p21 and rhoB p20), belonging to a ras p21/ras p21-like small molecular weight (Mr) GTP-binding protein (G protein) superfamily, was purified to near homogeneity from bovine brain cytosol and characterized. This regulatory protein, designated here as GDP dissociation inhibitor (GDI) for the rho proteins (rho GDI), inhibited the dissociation of GDP from rhoB p20 and the binding of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to the GDP-bound form of rhoB p20 but not of that to the guanine nucleotide-free form. The Mr value of rho GDI was estimated to be about 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and from the S value, indicating that rho GDI is composed of a single polypeptide without a subunit structure. The isoelectric point was about pH 5.7. rho GDI made a complex with the GDP-bound form of rhoB p20 with a molar ratio of 1:1 but not with the GTP gamma S-bound or guanine nucleotide-free form. rho GDI did not stimulate the GTPase activity of rhoB p20 and by itself showed neither GTP gamma S-binding nor GTPase activity. rho GDI was equally active for rhoA p21 and rhoB p20 but was inactive for other ras p21/ras p21-like G proteins including c-Ha-ras p21, smg p25A, and smg p21. rho GDI activity was detected in the cytosol fraction of various rat tissues. These results indicate that, in mammalian tissues, there is a novel type of regulatory protein specific for the rho proteins that interacts with the GDP-bound form of the rho proteins and thereby regulates the GDP/GTP exchange reaction of the rho proteins by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. Since there is a GTPase-activating protein for the rho proteins stimulating the GTPase activity of the rho proteins in mammalian tissues, the rho proteins appear to be regulated at least by GTPase-activating protein and GDI in a dual manner.     

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.     

RA-GEF, a novel Rap1A guanine nucleotide exchange factor containing a Ras/Rap1A-associating domain, is conserved between nematode and humans

A yeast two-hybrid screening for Ras-binding proteins in nematode Caenorhabditis elegans has identified a guanine nucleotide exchange factor (GEF) containing a Ras/Rap1A-associating (RA) domain, termed Ce-RA-GEF. Both Ce-RA-GEF and its human counterpart Hs-RA-GEF possessed a PSD-95/DlgA/ZO-1 (PDZ) domain and a Ras exchanger motif (REM) domain in addition to the RA and GEF domains. They also contained a region homologous to a cyclic nucleotide monophosphate-binding domain, which turned out to be incapable of binding cAMP or cGMP. Although the REM and GEF domains are conserved with other GEFs acting on Ras family small GTP-binding proteins, the RA and PDZ domains are unseen in any of them. Hs-RA-GEF exhibited not only a GTP-dependent binding activity to Rap1A at its RA domain but also an activity to stimulate GDP/GTP exchange of Rap1A both in vitro and in vivo at the segment containing its REM and GEF domains. However, it did not exhibit any binding or GEF activity toward Ras. On the other hand, Ce-RA-GEF associated with and stimulated GDP/GTP exchange of both Ras and Rap1A. These results indicate that Ce-RA-GEF and Hs-RA-GEF define a novel class of Rap1A GEF molecules, which are conserved through evolution.     

Liposomes comprising anionic but not neutral phospholipids cause dissociation of Rac(1 or 2) x RhoGDI complexes and support amphiphile-independent NADPH oxidase activation by such complexes

Activation of the phagocyte NADPH oxidase involves the assembly of a membrane-localized cytochrome b559 with the cytosolic components p47(phox), p67(phox), p40(phox), and the GTPase Rac (1 or 2). In resting phagocytes, Rac is found in the cytosol as a prenylated protein in the GDP-bound form, associated with the Rho GDP dissociation inhibitor (RhoGDI). In the process of NADPH oxidase activation, Rac is dissociated from RhoGDI and translocates to the membrane, in concert with the other cytosolic components. The mechanism responsible for dissociation of Rac from RhoGDI is poorly understood. We generated Rac(1 or 2) x RhoGDI complexes in vitro from recombinant Rac(1 or 2), prenylated enzymatically, and recombinant RhoGDI, and purified these by anion exchange chromatography. Exposing Rac(1 or 2)(GDP) x RhoGDI complexes to liposomes containing four different anionic phospholipids caused the dissociation of Rac(1 or 2)(GDP) from RhoGDI and its binding to the anionic liposomes. Rac2(GDP) x RhoGDI complexes were more resistant to dissociation, reflecting the lesser positive charge of Rac2. Liposomes consisting of neutral phospholipid did not cause dissociation of Rac(1 or 2) x RhoGDI complexes. Rac1 exchanged to the hydrolysis-resistant GTP analogue, GMPPNP, associated with RhoGDI with lower affinity than Rac1(GDP) and Rac1(GMPPNP) x RhoGDI complexes were more readily dissociated by anionic liposomes. Rac1(GMPPNP) x RhoGDI complexes elicited NADPH oxidase activation in native phagocyte membrane liposomes in the presence of p67(phox), without the need for an anionic amphiphile, as activator. Both Rac1(GDP) x RhoGDI and Rac1(GMPPNP) x RhoGDI complexes elicited amphiphile-independent, p67(phox)-dependent NADPH oxidase activation in phagocyte membrane liposomes enriched in anionic phospholipids but not in membrane liposomes enriched in neutral phospholipids.     

Sequential action of two GTPases to promote vacuole docking and fusion

Homotypic vacuole fusion occurs by sequential priming, docking and fusion reactions. Priming frees the HOPS complex (Vps 11, 16, 18, 33, 39 and 41) to activate Ypt7p for docking. Here we explore the roles of the GDP and GTP states of Ypt7p using Gdi1p (which extracts Ypt7:GDP), Gyp7p (a GTPase-activating protein for Ypt7p:GTP), GTPgammaS or GppNHp (non-hydrolyzable nucleotides), and mutant forms of Ypt7p that favor either GTP or GDP states. GDP-bound Ypt7p on isolated vacuoles can be extracted by Gdi1p, although only the GTP-bound state allows docking. Ypt7p is converted to the GTP-bound state after priming and stably associates with HOPS. Gyp7p can cause Ypt7p to hydrolyze bound GTP to GDP, driving HOPS release and accelerating Gdi1p-mediated release of Ypt7p. Ypt7p extraction does not inhibit the Ca(2+)-triggered cascade that leads to fusion. However, in the absence of Ypt7p, fusion is still sensitive to GTPgammaS and GppNHp, indicating that there is a second specific GTPase that regulates the calcium flux and hence fusion. Thus, two GTPases sequentially govern vacuole docking and fusion.     

Alantolactone promotes ER stress‐mediated apoptosis by inhibition of TrxR1 in triple‐negative breast cancer cell lines and in a mouse model

Triple‐negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and currently no effective targeted therapies are available. Alantolactone (ATL), a sesquiterpene lactone, has been shown to have potential anti‐tumour activity against various cancer cells. However, the underlying mechanism and therapeutic effect of ATL in the TNBC are largely unknown. In the present study, we found that ATL suppresses TNBC cell viability by reactive oxygen species (ROS) accumulation and subsequent ROS‐dependent endoplasmic reticulum (ER) stress both in vitro and in vivo. Thioredoxin reductase 1 (TrxR1) expression and activity of were significantly up‐regulated in the TNBC tissue specimens compare to the normal adjacent tissues. Further analyses showed that ATL inhibits the activity of TrxR1 both in vitro and in vivo in TNBC and knockdown of TrxR1 in TNBC cells sensitized ATL‐induced cell apoptosis and ROS increase. These results will provide pre‐clinical evidences that ATL could be a potential therapeutic agent against TNBC by promoting ROS‐ER stress‐mediated apoptosis through partly targeting TrxR1.