Conformational changes in the heat-denatured nucleotide complex of human lymphocytes on subsequent cooling

The microfluorometric technique allowed an assay of the chromatin state from binding of acridine orange in studies on the kinetics of conformational changes in the nucleoprotein complex (NP) of human peripheral blood lymphocytes. Extraction of F1 histone fraction with 0.01 N HCl (pH 2) substantially altered (decelerated) conformational changes in the NP complex. Extraction of F1, F2 and F3 histone fractions with 0.25N HCl (pH 0.6) led to a more pronounced deceleration of the kinetics of conformational changes. The latter ones were decelerated as well when the process was run in a more viscous medium (e. g. in a 70% glycerin solution). No conformational changes in the NP complex deproteinized with 0.25N HCl were revealed in a vicsous medium. In such a case the NP complex showed low F530 values and elevated values of the alpha coefficient that seems likely to suggest the nonreversible denaturation of the NP complex.     

Large Arf1 guanine nucleotide exchange factors: evolution,domain structure,and roles in membrane trafficking and human disease

The Sec7 domain ADP-ribosylation factor (Arf) guanine nucleotide exchange factors (GEFs) are found in all eukaryotes, and are involved in membrane remodeling processes throughout the cell. This review is focused on members of the GBF/Gea and BIG/Sec7 subfamilies of Arf GEFs, all of which use the class I Arf proteins (Arf1-3) as substrates, and play a fundamental role in trafficking in the endoplasmic reticulum (ER)—Golgi and endosomal membrane systems. Members of the GBF/Gea and BIG/Sec7 subfamilies are large proteins on the order of 200 kDa, and they possess multiple homology domains. Phylogenetic analyses indicate that both of these subfamilies of Arf GEFs have members in at least five out of the six eukaryotic supergroups, and hence were likely present very early in eukaryotic evolution. The homology domains of the large Arf1 GEFs play important functional roles, and are involved in interactions with numerous protein partners. The large Arf1 GEFs have been implicated in several human diseases. They are crucial host factors for the replication of several viral pathogens, including poliovirus, coxsackievirus, mouse hepatitis coronavirus, and hepatitis C virus. Mutations in the BIG2 Arf1 GEF have been linked to autosomal recessive periventricular heterotopia, a disorder of neuronal migration that leads to severe malformation of the cerebral cortex. Understanding the roles of the Arf1 GEFs in membrane dynamics is crucial to a full understanding of trafficking in the secretory and endosomal pathways, which in turn will provide essential insights into human diseases that arise from misregulation of these pathways.     

Nuclear functions of the Arf guanine nucleotide exchange factor BRAG2

BRAG2 is a guanine nucleotide exchange factor for the GTPase Arf6 that cycles between the cytoplasm and nucleus in a CRM1/exportin1-dependent manner. Despite its presence in the nucleus, nuclear functions have not previously been described. Here, we show that depletion of endogenous BRAG2 by RNAi leads to an increased number of Cajal bodies (CBs), and altered structure of nucleoli, as indicated by less focal fibrillarin staining. This result was surprising given that nuclear BRAG2 is diffusely distributed throughout the nucleoplasm and is not concentrated within nucleoli at steady state. However, we found that ectopic expression of the nuclear GTPase PIKE/AGAP2 causes both BRAG2 and the CB marker coilin to accumulate in nucleoli. Neither the GTPase activity of PIKE nor the nucleotide exchange activity of BRAG2 is required for this nucleolar concentration. Increased levels of exogenous BRAG2 in nucleoli result in a redistribution of fibrillarin to the nucleolar periphery, supporting a role for BRAG2 in regulating nucleolar architecture. These observations suggest that, in addition to its role in endocytic regulation at the plasma membrane, BRAG2 also functions within the nucleus.     

Identification of a guanine nucleotide exchange factor for Arf3, the yeast orthologue of mammalian Arf6

Small G proteins of the Arf and Rab families are fundamental to the organisation and activity of intracellular membranes. One of the most well characterised of these G proteins is mammalian Arf6, a protein that participates in many cellular processes including endocytosis, actin remodelling and cell adhesion. Exchange of GDP for GTP on Arf6 is performed by a variety of guanine nucleotide exchange factors (GEFs), principally of the cytohesin (PSCD) and EFA6 (PSD) families. In this paper we describe the characterisation of a GEF for the yeast orthologue of Arf6, Arf3, which we have named Yel1 (yeast EFA6-like-1) using yeast genetics, fluorescence microscopy and in vitro nucleotide exchange assays. Yel1 appears structurally related to the EFA6 family of GEFs, having an N-terminal Sec7 domain and C-terminal PH and coiled-coil domains. We find that Yel1 is constitutively targeted to regions of polarised growth in yeast, where it co-localises with Arf3. Moreover the Sec7 domain of Yel1 is required for its membrane targeting and for that of Arf3. Finally we show that the isolated Yel1 Sec7 domain strongly stimulates nucleotide exchange activity specifically on Arf3 in vitro.     

A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting the guanine nucleotide exchange factor GBF1

Many viruses modify cellular processes for their own benefit. The enterovirus 3A protein inhibits endoplasmic reticulum (ER)-to-Golgi transport, a function previously suggested to be important for viral suppression of immune responses. Here, we show that a virus carrying a 3A protein defective in inhibiting ER-to-Golgi transport is indeed less virulent in mice, and we unravel the mechanism by which 3A inhibits this trafficking step. Evidence is provided that 3A inhibits the activation of the GTPase ADP-ribosylation factor 1 (Arf1), which regulates the recruitment of the COP-I coat complex to membranes. 3A specifically inhibits the function of GBF1, a guanine nucleotide exchange factor for Arf1, by interacting with its N terminus. By specifically interfering with GBF1-mediated Arf1 activation, 3A may prove a valuable tool in dissecting the early steps of the secretory pathway.     

Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors

Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs (GBF and BIG) are found in all eukaryotes. In addition to the Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these noncatalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid, and cellular assays. Our data demonstrate a strong interaction between DCB domains within GBF1, BIG1, and BIG2 to maintain homodimers and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family and probably serves an important regulatory role in Arf activation.     

Regulation of Arf activation: the Sec7 family of guanine nucleotide exchange factors

The ADP ribosylation factors (Arfs) are a family of small, ubiquitously expressed and evolutionarily conserved guanosine triphosphatases that are key regulators of vesicular transport in eukaryotic cells (D'Souza-Schorey C, Chavrier P. ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 2006;7:347-358). Although Arfs are best known for their role in the nucleation of coat protein assembly at a variety of intracellular locations, it is increasingly apparent that they are also integral components in a number of important signaling pathways that are regulated by extracellular cues. The activation of Arfs is catalyzed by a family of guanine nucleotide exchange factors (GEFs), referred to as the Sec7 family, based on homology of their catalytic domains to the yeast Arf GEF, sec7p. While there are only six mammalian Arfs, the human genome encodes 15 Sec7 family members, which can be divided into five classes based on related domain organization. Some of this diversity arises from the tissue-specific expression of certain isoforms, but all mammalian cells appear to express at least six Arf GEFs, suggesting that Arf activation is under extensive regulatory control. Here we review recent progress in our understanding of the structure, localization and biology of the different classes of Arf GEFs.     

Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity

Arf6 is an isoform of Arf that localizes at the periphery of the cell where it has an essential role in endocytotic pathways. Its function does not overlap with that of Arf1, although the two proteins share approximately 70% sequence identity and they have switch regions, whose conformation depends on the nature of the guanine nucleotide, with almost identical sequences. The crystal structure of Arf6-GDP at 2.3 A shows that it has a conformation similar to that of Arf1-GDP, which cannot bind membranes with high affinity. Significantly, the switch regions of Arf6 deviate by 2-5 A from those of Arf1. These differences are a consequence of the shorter N-terminal linker of Arf6 and of discrete sequence changes between Arf6 and Arf1. Mutational analysis shows that one of the positions which differs between Arf1 and Arf6 affects the configuration of the nucleotide binding site and thus the nucleotide binding properties of the Arf variant. Altogether, our results provide a structural basis for understanding how Arf1 and Arf6 can be distinguished by their guanine nucleotide exchange factors and suggest a model for the nucleotide/membrane cycle of Arf6.     

Conformational changes associated with receptor-stimulated guanine nucleotide exchange in a heterotrimeric G-protein alpha-subunit: NMR analysis of GTPgammaS-bound states

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.     

Conformational states of the small G protein Arf-1 in complex with the guanine nucleotide exchange factor ARNO-Sec7

Arf1 is a small G protein involved in vesicular trafficking, and although it is only distantly related to Ras, it adopts a similar three-dimensional structure. In the present work, we study Arf1 bound to GDP and GTP and its interactions with one of its guanosine nucleotide exchange factors, ARNO-Sec7. The (31)P NMR spectra of Arf1.GDP.Mg(2+) and Arf1.GTP.Mg(2+) share the general features typical for all small G proteins studied so far. Especially, the beta-phosphate resonances of the bound nucleotide are shifted strongly downfield compared with the resonance positions of the free magnesium complexes of GDP and GTP. However, no evidence for an equilibrium between two conformational states of Arf1.GDP.Mg(2+) or Arf1.GTP.Mg(2+) could be observed as it was described earlier for Ras and Ran. Glu(156) of ARNO-Sec7 has been suggested to play as "glutamic acid finger" an important role in the nucleotide exchange mechanism. In the millimolar concentration range used in the NMR experiments, wild type ARNO-Sec7 and ARNO-Sec7(E156D) do weakly interact with Arf1.GDP.Mg(2+) but do not form a strong complex with magnesium-free Arf1.GDP. Only wild type ARNO-Sec7 competes weakly with GDP on Arf1.GDP.Mg(2+) and leads to a release of GDP when added to the solution. The catalytically inactive mutants ARNO-Sec7(E156A) and ARNO-Sec7(E156K) induce a release of magnesium from Arf1.GDP.Mg(2+) but do not promote GDP release. In addition, ARNO-Sec7 does not interact or only very weakly interacts with the GTP-bound form of Arf1, opposite to the observation made earlier for Ran, where the nucleotide exchange factor RCC1 forms a complex with Ran.GTP.Mg(2+) and is able to displace the bound GTP.     

Conformational changes in human lens proteins in cataract

The reactivity of protein thiol groups in human lens and the susceptibility of the proteins to tryptic digestion were investigated. Both were found to be greater in some cataractous lenses, indicating that lens proteins have unfolded during cataractogenesis. Almost all the tyrosine in the proteins of the normal human lens reacts with tetranitromethane and is therefore probably on the outside of the major lens proteins.     

SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses

SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both β-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.     

TRAPP stimulates guanine nucleotide exchange on Ypt1p

TRAPP, a novel complex that resides on early Golgi, mediates the targeting of ER-to-Golgi vesicles to the Golgi apparatus. Previous studies have shown that YPT1, which encodes the small GTP-binding protein that regulates membrane traffic at this stage of the secretory pathway, interacts genetically with BET3 and BET5. Bet3p and Bet5p are 2 of the 10 identified subunits of TRAPP. Here we show that TRAPP preferentially binds to the nucleotide-free form of Ypt1p. Mutants with defects in several TRAPP subunits are temperature-sensitive in their ability to displace GDP from Ypt1p. Furthermore, the purified TRAPP complex accelerates nucleotide exchange on Ypt1p. Our findings imply that Ypt1p, which is present on ER-to-Golgi transport vesicles, is activated at the Golgi once it interacts with TRAPP.     

Dynamics of GBF1, a Brefeldin A-sensitive Arf1 exchange factor at the Golgi

Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport. The mechanism of GBF1 binding to membranes is unknown. As a first step to understanding the mechanism of membrane association, we constructed a yellow fluorescent protein-tagged version of GBF1 and performed fluorescence recovery after photobleaching analysis to determine its residence time on Golgi membranes. We find that GBF1 molecules are not stably associated with the Golgi but rather cycle rapidly on and off membranes. The drug brefeldin A (BFA), an uncompetitive inhibitor of the exchange reaction that binds to an Arf-GDP-Arf GEF complex, stabilizes GBF1 on Golgi membranes. Using an in vivo assay to monitor Arf1-GTP levels, we show that GBF1 exchange activity on Arf1 is inhibited by BFA in mammalian cells. These results suggest that an Arf1-GBF1-BFA complex is formed and has a longer residence time on Golgi membranes than GBF1 or Arf1 alone.     

EFA6A,a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin‐1 and regulates neurite outgrowth

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6‐specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin‐1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two‐hybrid screening. The interaction was mediated by the C‐terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull‐down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post‐synaptic density and endosomes in dendritic spines. In Neuro‐2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co‐expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6‐mediated neurite formation through the interaction with SNX1.

     

Conformational changes in aspartate aminotransferase. Effect of active site ligands on peptide hydrogen-deuterium exchange

The conformational responses of aspartate aminotransferase (cytosolic isoenzyme from pig) to the binding of the coenzyme and competitive inhibitors and to the bond rearrangement steps during the transamination reaction were probed by the method of peptide hydrogen deuterium exchange. Binding of the coenzyme to the apoenzyme results in a marked retardation of hydrogen exchange; binding of the competitive inhibitor maleate to the pyridoxal enzyme induces a retardation of exchange somewhat exceeding that observed in the presence of the transaminating substrate pair glutamate and 2-oxoglutarate (Pfister, K., K?gi, J.H.R., and Christen, P. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 145-148). On formation of the complex of apoenzyme with N-(5'-phosphopyridoxyl)-L-glutamate or-L-aspartate, analogs of the covalent coenzyme substrate intermediates, a similar exchange retardation occurs. The extent of the exchange retardation in these different functional states of the enzyme correlates with previous results of differential chemical and proteolytic modifications. Apparently, the diverse methods register shifts in one and the same conformational equilibrium. Moreover, the conditions under which peptide hydrogen exchange indicates a pronounced tightening of the protein matrix correspond with those inducing crystallization of the enzyme in the "closed" form. Thus, the transition between the "open" and "closed" form of the enzyme, i.e. the bulk movement of the small domain, as observed and defined by x-ray crystallography (Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H., and Christen, P. (1984) J. Mol. Biol. 174, 497-525) is the major structural correlate of the conformational changes undergone by the enzyme in solution.     

Modeling the dynamic behaviors of the COPI vesicle formation regulators,the small GTPase Arf1 and its activating Sec7 guanine nucleotide exchange factor GBF1 on Golgi membranes

The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain–containing guanine nucleotide exchange factor GBF1 (Golgi brefeldin A resistant guanine nucleotide exchange factor 1). This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have atomic-level knowledge of Arf1 activation by Sec7 domain–containing GEFs, our understanding of the biophysical processes regulating Arf1 and GBF1 dynamics is limited. We used fluorescence recovery after photobleaching data and kinetic Monte Carlo simulation to assess the behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses suggest that Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1. Furthermore, the GBF1-mediated Arf1 activation is much faster than GBF1 cycling on/off the membrane, suggesting that GBF1 is regulated by processes other than its interactions Arf1. Interestingly, modeling the behavior of the catalytically inactive GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1 and suggests that GBF1/E794K is stabilized on the membrane independently of complex formation.