Isolation and Analysis of Mitochondrial Fission Enzyme DNM1 from Saccharomyces cerevisiae.

Mitochondrial fission, an essential process for mitochondrial and cellular homeostasis, is accomplished by evolutionarily conserved members of the dynamin superfamily of large GTPases. These enzymes couple the hydrolysis of guanosine triphosphate to the mechanical work of membrane remodeling that ultimately leads to membrane scission. The importance of mitochondrial dynamins is exemplified by mutations in the human family member that causes neonatal lethality. In this chapter, we describe the subcloning, purification, and preliminary characterization of the budding yeast mitochondrial dynamin, DNM1, from Saccharomyces cerevisiae, which is the first mitochondrial dynamin isolated from native sources. The yeast-purified enzyme exhibits assembly-stimulated hydrolysis of GTP similar to other fission dynamins, but differs from the enzyme isolated from non-native sources.

Purification and Characterization of MxB.

MxB/Mx2 is an interferon-induced dynamin-like GTPase, which restricts a number of life-threatening viruses. Because of its N-terminal region, predicted to be intrinsically disordered, and its propensity to self-oligomerize, purification of the full-length protein has not been successful in conventional E. coli expression systems. In this chapter, we describe an expression and purification procedure to obtain pure full-length wild-type MxB from suspension-adapted mammalian cells. We further describe how to characterize its GTPase activity and oligomerization function.

Affinity Purification and Functional Characterization of Dynamin-Related Protein 1.

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Analysis of Mitochondrial Membrane Fusion GTPase OPA1 Expressed by the Silkworm Expression System.

Mitochondria are highly dynamic organelles, which move and fuse to regulate their shape, size, and fundamental function. The dynamin-related GTPases play a critical role in mitochondrial membrane fusion. In vitro reconstitution of membrane fusion using recombinant proteins and model membranes is quite useful in elucidating the molecular mechanisms underlying membrane fusion and to identify the essential elements involved in fusion. However, only a few reconstituting approaches have been reported for mitochondrial fusion machinery due to the difficulty of preparing active recombinant mitochondrial fusion GTPases. Recently, we succeeded in preparing a sufficient amount of recombinant OPA1 involved in mitochondrial inner membrane fusion using a BmNPV bacmid-silkworm expression system. In this chapter, we describe the method for the expression and purification of a membrane-anchored form of OPA1 and liposome-based in vitro reconstitution of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays.

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB).

Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 µL assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a~2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.

Insights into the GTP/GDP cycle of RabX3, a novel GTPase from Entamoeba histolytica with tandem G-domains.

Members of the small GTPase Ras superfamily regulate a host of systems through their ability to catalyze the GTP/GDP cycle. All family members reported thus far possess a single GTPase domain with a P-loop containing a nucleoside triphosphate hydrolase fold. Here for the first time we report a novel member from Entamoeba histolytica, EhRabX3, which harbors two GTPase domains in tandem and exhibits unique biochemical properties. A combination of biochemical and microcalorimetric studies revealed that EhRabX3 binds to a single guanine nucleotide through its N-terminal domain. Unlike most of the members of the Ras superfamily, the dissociation of the nucleotide from EhRabX3 is independent of Mg(2+), perhaps indicating a novel mechanism of nucleotide exchange by this protein. We found that EhRabX3 is extremely sluggish in hydrolyzing GTP, and that could be attributed to its atypical nucleotide binding pocket. It harbors substitutions at two positions that confer oncogenicity to Ras because of impaired GTP hydrolysis. Engineering these residues into the conserved counterparts enhanced their GTPase activity by at least 20-fold. In contrast to most of the members of the Ras superfamily, EhRabX3 lacks the prenylation motif. Using indirect immunofluorescence and biochemical fractionation, we demonstrated that the protein is distributed all over the cytosol in amoebic trophozoites. Collectively, this unique ancient GTPase exhibits a striking evolutionary divergence from the other members of the superfamily.

Binding to G-quadruplex RNA activates the mitochondrial GTPase NOA1.

NOA1 is an evolutionary conserved, nuclear encoded GTPase essential for mitochondrial function and cellular survival. The function of NOA1 for assembly of mitochondrial ribosomes and regulation of OXPHOS activity depends on its GTPase activity, but so far no ligands have been identified that regulate the GTPase activity of NOA1. To identify nucleic acids that bind to the RNA-binding domain of NOA1 we employed SELEX (Systemic Evolution of Ligands by EXponential Enrichment) using recombinant mouse wildtype NOA1 and the GTPase mutant NOA1-K353R. We found that NOA1 binds specifically to oligonucleotides that fold into guanine tetrads (G-quadruplexes). Binding of G-quadruplex oligonucleotides stimulated the GTPase activity of NOA1 suggesting a regulatory link between G-quadruplex containing RNAs, NOA1 function and assembly of mitochondrial ribosomes.

Purification, crystallization and X-ray diffraction analysis of human dynamin-related protein 1 GTPase-GED fusion protein.

The mechano-enzyme dynamin-related protein 1 plays an important role in mitochondrial fission and is implicated in cell physiology. Dysregulation of Drp1 is associated with abnormal mitochondrial dynamics and neuronal damage. Drp1 shares structural and functional similarities with dynamin 1 with respect to domain organization, ability to self-assemble into spiral-like oligomers and GTP-cycle-dependent membrane scission. Structural studies of human dynamin-1 have greatly improved the understanding of this prototypical member of the dynamin superfamily. However, high-resolution structural information for full-length human Drp1 covering the GTPase domain, the middle domain and the GTPase effector domain (GED) is still lacking. In order to obtain mechanistic insights into the catalytic activity, a nucleotide-free GTPase-GED fusion protein of human Drp1 was expressed, purified and crystallized. Initial X-ray diffraction experiments yielded data to 2.67 Šresolution. The hexagonal-shaped crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 53.59, b = 151.65, c = 43.53 Å, one molecule per asymmetric unit and a solvent content of 42%. Expression of selenomethionine-labelled protein is currently in progress. Here, the expression, purification, crystallization and X-ray diffraction analysis of the Drp1 GTPase-GED fusion protein are presented, which form a basis for more detailed structural and biophysical analysis.

Human C4orf14 interacts with the mitochondrial nucleoid and is involved in the biogenesis of the small mitochondrial ribosomal subunit.

The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.

Purification, crystallization and preliminary X-ray crystallographic analysis of Arabidopsis thaliana dynamin-related protein 1A GTPase-GED fusion protein.

Plant-specific dynamin-related proteins play crucial roles in cell-plate formation, endocytosis or exocytosis, protein sorting to the vacuole and plasma membrane and the division of mitochondria and chloroplasts. In order to determine the crystal structure and thus to obtain a better understanding of the biological functions and mechanisms of dynamin-related proteins in plant cells, the GTPase domain of Arabidopsis thaliana dynamin-related protein 1A (AtDRP1A) fused to its GTPase effector domain (GED) was crystallized in a nucleotide-associated form using polyethylene glycol 3350 as precipitant. The hexagonal crystals (space group P6(1)) had unit-cell parameters a = b = 146.2, c = 204.3 Å, and diffraction data were collected to 3.6 Å resolution using synchrotron radiation. Four molecules, comprising two functional dimers, are assumed per asymmetric unit, corresponding to a Matthews coefficient of 3.9 Å(3) Da(-1) according to the molecular weight of 39 kDa.

Study on the chaperone properties of conserved GTPases.

As a large family of hydrolases, GTPases are widespread in cells and play the very important biological function of hydrolyzing GTP into GDP and inorganic phosphate through binding with it. GTPases are involved in cell cycle regulation, protein synthesis, and protein transportation. Chaperones can facilitate the folding or refolding of nascent peptides and denatured proteins to their native states. However, chaperones do not occur in the native structures in which they can perform their normal biological functions. In the current study, the chaperone activity of the conserved GTPases of Escherichia coli is tested by the chemical denaturation and chaperone-assisted renaturation of citrate synthase and α-glucosidase. The effects of ribosomes and nucleotides on the chaperone activity are also examined. Our data indicate that these conserved GTPases have chaperone properties, and may be ancestral protein folding factors that have appeared before dedicated chaperones.

A cold-active heat-labile t-RNA modification GTPase from a psychrophilic bacterium Pseudomonas syringae (Lz4W).

A cold-active heat-labile t-RNA modification GTPase (TrmE) from psychrophilic bacterium Pseudomonas syringae (Lz4W) has been purified and characterized. The purified TrmE is a 53 kDa protein, has GTPase activity and hydrolyses only the oxy and deoxy forms of GTP but not the other nucleotide triphosphates. The enzyme exhibits optimal activity at 12-18 degrees C and retains 65% of its optimal activity at 4 degrees C, indicating that it is a cold-active enzyme. The enzyme is also heat-labile and loses 60% of its activity at 30 degrees C. This is the first report on the purification and characterization of a TrmE from a psychrophilic bacterium.

GDP-capture compound--a novel tool for the profiling of GTPases in pro- and eukaryotes by capture compound mass spectrometry (CCMS).

The functional isolation of proteome subsets based on small molecule-protein interactions is an increasingly popular and promising field in functional proteomics. Entire protein families may be profiled on the basis of their common interaction with a metabolite or small molecule inhibitor. This is enabled by novel multifunctional small molecule probes. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate Capture Compound-protein conjugates from complex biological samples for direct trypsinisation and protein identification by liquid chromatography/mass spectrometry (CCMS). We here present the synthesis and application of a novel GDP-Capture Compound for the functional enrichment of GTPases, a pivotal protein family that exerts key functions in signal transduction. We present data from CCMS experiments on two biological lysates from Escherichia coli and from human-derived Hek293 cells. The GDP-Capture Compound robustly captures a wide range of different GTPases from both systems and will be a valuable tool for the proteomic profiling of this important protein family.

Functional characterization of phosphorylation sites in dynamin-related protein 1.

Dynamin-related protein 1 is member of the dynamin-family of large GTPases. Similar to the endocytosis motor dynamin, Drp1 uses GTP hydrolysis to power constriction of the outer mitochondrial membrane and ultimately mitochondrial division. Dynamin phosphorylation in its unique C-terminal proline-rich domain interferes with binding of accessory proteins that induce membrane curvature and inhibits clathrin-mediated endocytosis. Evidence within the last few years indicates that Drp1 is also regulated by the phosphorylation/dephosphorylation cycle. Drp1 regulation is complex, in that both inhibitory and activating phosphorylations have been described that lead to, respectively, mitochondrial elongation and shortening. In this chapter, we describe methods for the identification and functional characterization of Drp1 phosphorylation sites. Among these methods is replacement of the endogenous protein by phosphorylation-site mutant Drp1 via combined shRNA and RNAi-resistant cDNA expression from the same plasmid. We also discuss primary astrocyte cultures as a model for regulation of cell death and mitochondrial morphology by Drp1 and present ImageJ macro source code for unbiased quantification of mitochondrial shape changes.

Properties of HflX, an enigmatic protein from Escherichia coli.

The Escherichia coli gene hflX was first identified as part of the hflA operon, mutations in which led to an increased frequency of lysogenization upon infection of the bacterium by the temperate coliphage lambda. Independent mutational studies have also indicated that the HflX protein has a role in transposition. Based on the sequence of its gene, HflX is predicted to be a GTP-binding protein, very likely a GTPase. We report here purification and characterization of the HflX protein. We also specifically examined its suggested functional roles mentioned above. Our results show that HflX is a monomeric protein with a high (30% to 40%) content of helices. It exhibits GTPase as well as ATPase activities, but it has no role in lambda lysogeny or in transposition.

Crystallization and preliminary X-ray crystallographic analysis of the probable tRNA-modification GTPase (TrmE) from Staphylococcus aureus.

Probable tRNA-modification GTPase (TrmE) is a guanine nucleotide-binding protein that is conserved between bacteria and humans. GTPase hydrolyzes GTP and plays a pivotal role in signalling pathways. In this study, TrmE from Staphylococcus aureus was overexpressed in Escherichia coli. The enzyme was found to crystallize at 295 K when ammonium sulfate was used as a precipitant. X-ray diffraction data were collected to 2.9 A resolution from the crystallized enzyme using synchrotron radiation. The crystal was found to belong to the cubic space group I23, with unit-cell parameters a = b = c = 229.47 A, alpha = beta = gamma = 90 degrees . The crystal is likely to contain four monomers in the asymmetric unit, with a corresponding V(M) of 2.4 A(3) Da(-1) and a solvent content of 50%.

Biochemical characterization of membrane-associated septin from rat brain.

Within the cell membrane there exist various microdomains (lipid rafts) in which specific lipids and proteins are assembled and these microdomains are recovered in the detergent-resistant low-density membrane fraction (DRM). Septin is a novel GTP-binding, cytoskeletal protein having various isoforms that assemble into homo- and heterooligomers and filaments. As the localization of septin 3 in DRM was found through a proteomics analysis of brain-derived DRM, the presence of other septin isoforms in DRM was studied. Western blotting analysis showed maturation-dependent enrichment of several septin isoforms in DRM prepared from synaptic plasma membrane (SPM). These isoforms were solubilized with high MgCl2 solution and recovered as the precipitate after dialysis to low ionic solution. Three times cycling of the extraction-dialysis process resulted in the partial purification of septin complex and electron microscopic observation of this fraction revealed rod-like structures in which building units were observed. The presence of heterooligomers was shown with western blotting after the separation of the MgCl2 extract with blue-native polyacrylamide gel electrophoresis. Immunoprecipitation assay using monoclonal anti-septin11 antibody also showed the presence of heterooligomers. These results show that septin localizes in the membrane microdomains of the SPM in adult brain and may have important roles in the membrane dynamics of neurons.

Characterization of OPA1 isoforms isolated from mouse tissues.

OPA1, a nuclear encoded mitochondrial protein causing autosomal dominant optic atrophy, is a key player in mitochondrial fusion and cristae morphology regulation. In the present study, we have compared the OPA1 transcription and translation products of different mouse tissues. Unlike in humans, we found only two exons (4b and 5b) to be involved in alternative splicing. The relative abundance of the resulting four different splice variants is tissue-dependent. Proteolytic cleavage by mitochondrial processing peptidase generates two long forms, isoforms 1 and 7, which lead to three short forms representing the end products after further proteolytic processing. In contrast, isoforms 5 and 8 are directly processed into their corresponding short forms. Short form 1 molecules form 184 kDa dimers, whereas all other isoforms contribute to 285 kDa complexes. Coiled-coil domains of the OPA1 protein specifically homo-associate and may be involved in the formation of these complexes. Furthermore, the region encoded by exon 5b inhibits the self-association of coiled-coil domain-I. Finally, our data pinpoint isoform 1 as the, by far, most abundant isoform in the nervous tissue. We postulate that manipulation of isoform 1 protein levels in relation to the other isoforms induces changes in the mitochondrial network in the cell and therefore, mutations affecting the level of functional isoform 1 could lead to devastating effects on retinal ganglion cells.