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• W2000224269 abstract "Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction. Filament formation is required for most of the functions of actin. However, the intermonomer interactions that stabilize F-actin have not been elucidated because of a lack of an F-actin crystal structure. The Holmes muscle actin model suggests that an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the same strand contributes to this stabilization. Yeast actin has an Ala-167 instead. F-actin molecular dynamics modeling predicts another interaction between Arg-39 of one monomer and Asp-275 of an opposing strand monomer. In Toxoplasma gondii actin, which forms short stubby filaments, the Asp-275 equivalent is replaced by Arg leading to a potential filament-destabilizing charge-charge repulsion. Using yeast actin, we tested the effect of A167E as a potential stabilizer and A167R and D275R as potential filament disruptors. All mutations caused abnormal growth and mitochondrial malfunction. A167E and D275R actins polymerize normally and form relatively normal appearing filaments. A167R nucleates filaments more slowly and forms filament bundles. The R39D/A167R double mutant, which re-establishes an ionic bond in the opposite orientation, reverses this polymerization and bundling defect. Stoichiometric amounts of yeast cofilin have little effect on wild-type and A167E filaments. However, D275R and A167R actin depolymerization is profound with cofilin. Although our results suggest that disruption of an interaction between Arg-39 and Asp-275 is not sufficient to cause fragmentation, it suggests that it changes filament stability thereby disposing it for enhanced cofilin depolymerizing effects. Ala-167 results demonstrate the in vivo and in vitro importance of another potential Arg-39 ionic interaction. Actin is a 42-kDa eukaryotic protein highly conserved from yeast to humans. It is involved in many physiological processes such as cell motility, establishment of polarity, contraction, cytokinesis, intracellular trafficking, and maintenance of structural integrity. In physiological conditions, monomeric G-actin will polymerize to filamentous F-actin, and this ability to form a double-stranded filament is required for actin to carry out the vast majority of its physiological functions (1.Carlier M.F. Pantaloni D. J. Biol. Chem. 2007; 282: 23005-23009Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 2.Kaksonen M. Toret C.P. Drubin D.G. Nat. Rev. Mol. Cell Biol. 2006; 7: 404-414Crossref PubMed Scopus (568) Google Scholar, 3.Moseley J.B. Goode B.L. Microbiol. Mol. Biol. Rev. 2006; 70: 605-645Crossref PubMed Scopus (286) Google Scholar). X-ray crystallography (4.Kabsch W. Mannherz H.G. Suck D. Pai E.F. Holmes K.C. Nature. 1990; 347: 37-44Crossref PubMed Scopus (1546) Google Scholar, 5.McLaughlin P.J. Gooch J.T. Mannherz H.G. Weeds A.G. Nature. 1993; 364: 685-692Crossref PubMed Scopus (498) Google Scholar, 6.Chik J.K. Lindberg U. Schutt C.E. J. Mol. Biol. 1996; 263: 607-623Crossref PubMed Scopus (185) Google Scholar, 7.Morton W.M. Ayscough K.R. McLaughlin P.J. Nat. Cell Biol. 2000; 2: 376-378Crossref PubMed Scopus (391) Google Scholar, 8.Klenchin V.A. Allingham J.S. King R. Tanaka J. Marriott G. Rayment I. Nat. Struct. Biol. 2003; 10: 1058-1063Crossref PubMed Scopus (128) Google Scholar, 9.Otterbein L.R. Graceffa P. Dominguez R. Science. 2001; 293: 708-711Crossref PubMed Scopus (417) Google Scholar, 10.Graceffa P. Dominguez R. J. Biol. Chem. 2003; 278: 34172-34180Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) shows that G-actin has four subdomains, and it binds nucleotide, ATP or ADP, and divalent cation in the cleft between subdomains 2 and 4. Although G-actin has a slight ATPase activity, this activity is enhanced several fold in F-actin (11.Pollard T.D. Weeds A.G. FEBS Lett. 1984; 170: 94-98Crossref PubMed Scopus (89) Google Scholar). Although several high resolution structures of G-actin have been solved, there are only models for the F-actin filament. Currently, the most accepted models of F-actin are derivatives of the Holmes model generated by fitting the monomer structure of actin in the closed state to a low electron density map of oriented F-actin gels (12.Lorenz M. Poole K.J. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar, 13.Holmes K.C. Popp D. Gebhard W. Kabsch W. Nature. 1990; 347: 44-49Crossref PubMed Scopus (1325) Google Scholar). The contacts between the monomers are made in a way that subdomains 1 and 2 largely constitute the outside surface and subdomains 3 and 4 the internal strand-strand interface. However, the precise intermonomer interactions that lead to the stabilization of this filament structure have not been elucidated. One of the major ways that intermonomer contacts in the filament might be stabilized is via intermonomer ionic interactions. Modeling studies have suggested that two ionic bonds, each involving Arg-39 in the DNase I loop in subdomain 2 of one monomer, might play such a role. The original Holmes filament model of muscle actin predicts an ionic interaction between Arg-39 of one monomer and Glu-167 of an adjacent monomer in the opposing strand of the actin filament (12.Lorenz M. Poole K.J. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar). This same interaction would also be present in mammalian β- and γ-nonmuscle actins. Yeast actin is 87% homologous with muscle actin and 90% homologous with the two nonmuscle actins, making it an excellent model for understanding basic actin biochemical behavior. In yeast actin, Glu-167 is replaced by an Ala, preventing such an interaction from occurring. Yeast actin filaments fragment more easily than do muscle actin filaments (14.Buzan J.M. Frieden C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 91-95Crossref PubMed Scopus (62) Google Scholar) and show a commensurate shorter mean filament length (9 μm for muscle (15.Isambert H. Venier P. Maggs A.C. Fattoum A. Kassab R. Pantaloni D. Carlier M.F. J. Biol. Chem. 1995; 270: 11437-11444Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar) compared with 3.6 μm for yeast actin (16.McKane M. Wen K.K. Meyer A. Rubenstein P.A. J. Biol. Chem. 2006; 281: 29916-29928Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar)) suggesting that yeast actin filaments are less stable than muscle filaments. This change correlates with a decreased ionic interaction resulting from the Glu-167 to Ala change, but the existence of this ionic interaction or its importance to actin filament stability has not been experimentally addressed. Suggestion of a second potential filament-stabilizing ionic interaction grew out of the work of Sahoo et al. (17.Sahoo N. Beatty W. Heuser J. Sept D. Sibley L.D. Mol. Biol. Cell. 2006; 17: 895-906Crossref PubMed Google Scholar) on the properties of actin from the parasite Toxoplasma gondii. Molecular dynamics modeling of muscle actin predicted an ionic interaction between Arg-39 of one monomer and a Glu at residue 275 of a neighboring monomer, which is not predicted by the Holmes model. In yeast actin, this Glu is replaced by an Asp, still permitting the formation of this ionic bond. T. gondii uses short actin filaments for a gliding type of motility and host invasion, and purified T. gondii actin will not form normal-appearing long filaments in vitro. The residue in T. gondii actin that corresponds to 275 in muscle actin is Arg-276. The arginine here would create a repulsive interaction between Arg-276 and Arg-39 of adjacent monomers potentially leading to filament destabilization and perhaps resulting in the short filaments that were observed. Again, the effect of this repulsive interaction alone on actin filament stability has not been tested. Positions of the residues in question in the Holmes filament model are represented in Fig. 1. The Arg-39 to Glu-167 bond distance is 4.02 Å (12.Lorenz M. Poole K.J. Popp D. Rosenbaum G. Holmes K.C. J. Mol. Biol. 1995; 246: 108-119Crossref PubMed Scopus (192) Google Scholar), and the Arg-39 to Asp-275 bond distance is between 3 and 3.5 Å (17.Sahoo N. Beatty W. Heuser J. Sept D. Sibley L.D. Mol. Biol. Cell. 2006; 17: 895-906Crossref PubMed Google Scholar). 2D. Sept, personal communication. Predictions based on modeling studies require experimental studies to establish whether they are correct or incorrect. In this study, we have used site-directed mutagenesis of yeast actin to introduce mutations at residues 167 and 275 that might be expected to lead either to ionic bond stabilization or destabilization with Arg-39. We then examine the effects of these mutations in vivo in yeast, purified the mutant actins, and assessed the effects of these mutations on actin polymerization in vitro. DNase I (grade D) was purchased from Worthington Biochemicals. DE52 DEAE-cellulose was obtained from Whatman. The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA), and the DNA primers used for site-directed mutagenesis were obtained from Integrated DNA Technologies (Coralville, IA). Rhodamine-phalloidin, FM 4–64, and 4′,6-diamidino-2-phenylindole were purchased from Molecular Probes-Invitrogen. 1,N6-Ethenoadenosine 5′-triphosphate (ϵ-ATP) 3The abbreviations used are: ϵ-ATP, 1,N6-ethenoadenosine 5′-triphosphate; GFP, green fluorescent protein; WT, wild type; EM, electron microscopy. was purchased from Invitrogen. Yeast cakes for wild-type (WT) yeast actin controls were purchased from a local bakery. All other chemicals used were of reagent grade quality. Site-directed mutagenesis was performed using a Stratagene kit according to the manufacturer's instruction. The template plasmid was pRS314-based, which is a TRP1-marked yeast shuttle vector into which was inserted the yeast ACT1 promoter and coding sequence (18.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). Plasmids that had the desired actin mutation were transformed into a yeast strain whose chromosomal actin gene was disrupted by the LEU2 gene. In this strain, WT actin was expressed from another centromeric plasmid marked with the URA3 gene. Cells were grown in the appropriate selection media to select for the plasmid carrying the mutated actin gene and to encourage elimination of the plasmid carrying the WT actin gene (16.McKane M. Wen K.K. Meyer A. Rubenstein P.A. J. Biol. Chem. 2006; 281: 29916-29928Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Plasmids were isolated from the final colonies and sequenced to ensure the presence of the desired mutation. R39D/A167R actin could not be generated from a normal WT cell host, because it was not compatible with cell viability. However, it was successfully obtained spontaneously as a suppressor from a yeast strain carrying a mutant profilin (details to be published elsewhere). To examine the effects of the mutations on growth characteristics, growth curves were generated. WT and mutant strains were grown in YPD (1% yeast extract, 2% peptone, and 2% dextrose) liquid media at 30 °C. Cell density (A600) was monitored with time, and doubling times were determined as described previously (19.McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Temperature sensitivity, hyperosmotic sensitivity, and growth on glycerol were examined as described previously (19.McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). A PCR fragment was generated from the plasmid pFA6a-GFP-kanMX6 that has the KAN gene in the middle and AIP1 homologous sequence on the ends. The primers used for the PCR were: forward: 5′-agggcgaagactctgcgttgtgggagaaggtcgtgataactaacagctgctgggattacacatgg-3′ and reverse: 5′-cctcttcaatttcctcttcgtttgctcccttttcagcaggacttcgcatctgggcagatgatgtc-3′. The sequences in bold are homologous to the AIP1 gene, whereas the rest of the primers are homologous to the plasmid. PCR was performed using an Invitrogen Platinum® TaqDNA Polymerase High Fidelity kit with 30 cycles of 30 s at 94 °C, 30 s at 55 °C, and 3 min at 68 °C to generate an ∼1500-bp fragment. Yield was approximated by visualization on an agarose gel. pCENWT was transformed with 500 ng of fragment via lithium acetate, and transformants were selected on YPD Geneticin plates. pCENWT strain is a trp1,ura3–52 haploid cell in which the chromosomal ACT1 gene has been disrupted by replacement of the coding sequence with the LEU2 gene, and the WT actin gene and its promoter are on a centromeric plasmid containing the URA3 gene. Homologous recombination of the fragment into the chromosome deleted ∼1200 bp of the AIP1 gene. Colonies were patched on YPD, and correct insertion into the chromosome was confirmed by PCR of the genomic DNA using a forward primer homologous to sequence upstream from AIP1 and a reverse primer homologous to the KAN gene. Conditions for PCR were the same as above, and the primers used were: forward: 5′-tggtcatggctcttcggtagtcac-3′ and reverse: 5′-gtattctgggcctccatgtcgctgg-3′. Using the pCENWT Δaip1 strain as the host, WT yeast actin or the various mutant actins housed on pRS314 plasmids, which contain the coding sequence for the actin and promoter region in addition to the TRP1 gene, were transformed into this haploid yeast strain by lithium acetate. Transformants were selected on tryptophan-deficient medium and then subjected to plasmid shuffling to eliminate the WT actin gene (16.McKane M. Wen K.K. Meyer A. Rubenstein P.A. J. Biol. Chem. 2006; 281: 29916-29928Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Mutant actins were purified from lysates of the mutant yeast cells in a procedure that includes DNase I affinity chromatography and DE52 DEAE-cellulose chromatography (20.Cook R.K. Blake W.T. Rubenstein P.A. J. Biol. Chem. 1992; 267: 9430-9436Abstract Full Text PDF PubMed Google Scholar). After purification from cell lysates, actin was subjected to a polymerization/depolymerization step, including centrifugation in a Beckman TLA 100.2 rotor at 80,000 rpm for 1 h to remove denatured protein and actin oligomers, which might otherwise act as nuclei. The purified actins, stored at 4 °C in G-buffer (10 mm Tris-HCl, pH 7.5, 0.2 mm CaCl2, 0.2 mm ATP), were used within 4 days. For each experiment, WT yeast actin was also prepared as a purification and experimental control. Yeast cofilin was purified as described previously (21.Ojala P.J. Paavilainen V. Lappalainen P. Biochemistry. 2001; 40: 15562-15569Crossref PubMed Scopus (69) Google Scholar). Thermal Denaturation—The apparent melting temperatures for WT and mutant actins were determined by CD as described previously (22.Chen X. Cook R.K. Rubenstein P.A. J. Cell Biol. 1993; 123: 1185-1195Crossref PubMed Scopus (88) Google Scholar). Briefly, the ellipticity of sample of 1 μm actin in G-buffer was monitored at 222 nm in an AVIV 62 DS spectropolarimeter as the sample was heated at a constant rate of l°C/min over a temperature range extending from 25 °C to 85 °C. Data were fitted to a two-state model, and the apparent Tm value was determined by fitting the data to the Gibbs-Helmholtz equation to approximate the temperature at which 50% of the actin was denatured. Nucleotide Exchange—To determine the half time of the nucleotide exchange ϵ-ATP was used as described previously (23.Yao X. Nguyen V. Wriggers W. Rubenstein P.A. J. Biol. Chem. 2002; 277: 22875-22882Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). Excess ATP was removed from 3 μm samples of G-actin with Micro Bio-Spin 6 columns (Bio-Rad). The samples were then incubated with ϵ-ATP at a final concentration 1.25 mm for 2 h at 4 °C. After incubation, excess ϵ-ATP was removed with Micro Bio-Spin 6 columns (Bio-Rad). The decrease of the ϵ-ATP fluorescence signal was monitored after addition of 100 μm ATP in a Fluorolog fluorescence spectrometer (HORIBA Jobin Yvon Inc.) with excitation set at 340 nm and emission set at 410 nm. Exchange half times were determined by fitting the data with BioKine Version 3.1 (Bio Logic, France). Binding of G-actin to Cofilin—G-actin samples were labeled with N-(1-pyrenyl)maleimide (Sigma) for 2 h at 4 °C. N-(1-Pyrenyl)maleimide was added in a slight excess so that final concentration is 1.1 pyrene to 1 actin. Before labeling dithiothreitol was removed with Micro Bio-Spin 6 columns. After labeling, excess pyrene was removed with Micro Bio-Spin 6 columns. Cofilin was titrated into samples of 1 μm pyrene-labeled G-actin, and the increase in pyrene signal was monitored with a Fluorolog fluorescence spectrometer (HORIBA Jobin Yvon Inc.) with excitation set at 344 nm and emission set at 365 nm. The experiments were done in G-buffer that contained 50 mm KCl. The binding data were analyzed in Excel and fitted to Equation 1, f=fi[A]+[C]+Kd—([A]+[C]+Kd)2—4[A][C]2(Eq. 1) where f is the observed fluorescence minus the fluorescence of G-actin alone, fi is the molar fluorescence intensity obtained with best fit in Excel, [A] is the actin concentration, [C] is the cofilin concentration, and Kd is the dissociation constant. Actin Polymerization Assays—Polymerization was induced by the addition of F-salts, 2 mm MgCl2 and 50 mm KCl, to a 5 μm G-actin sample in a total volume of 120 μl. Polymerization was monitored at 25 °C by following the increase in light scattering of the sample in a thermostatted microcuvette in a Fluoro-Max-3 fluorescence spectrometer (HORIBA Jobin Yvon Inc.) with both excitation and emission wavelengths set at 360 nm. Electron Microscopy and Actin Filament Length Measurement—For actin filament visualization, 2 μl of samples of 5 μm F-actin was deposited on carbon-coated Formvar grids and negatively stained with 1% uranyl acetate. Samples were observed with a JEOL JEM-1230 transmission electron microscope at the University of Iowa Central Microscopy Research Facilities, and images were recorded with a Gatan UltraScan 1000 2 × 2k charge-coupled device camera. For each sample, the lengths of more than 100 filaments were determined using NIH ImageJ software. Phosphate Release Assay—EnzChek® Phosphate Assay Kit from Molecular Probes was used as described previously (24.Yao X. Rubenstein P.A. J. Biol. Chem. 2001; 276: 25598-25604Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). To determine if mutant F-actins bound cofilin, a cosedimentation assay with few modifications was used (25.Rodal A.A. Tetreault J.W. Lappalainen P. Drubin D.G. Amberg D.C. J. Cell Biol. 1999; 145: 1251-1264Crossref PubMed Scopus (178) Google Scholar). G-actin was centrifuged for 30 min at 80,000 rpm at 4 °C in a TLA 100.2 rotor (Beckman Instruments) to remove residual actin filament seeds. 60 μl of 5 μm G-actin was polymerized with the addition of F-salts. After 30 min, when polymerization had reached steady state, cofilin was added at an actin:cofilin ratio of 1:1. After 10 min the samples were centrifuged in a TLA 100 rotor for 20 min at 80,000 rpm at 25 °C, conditions sufficient to pellet F-actin. Pellets were resuspended in 60 μl of F-buffer, and equal amounts of supernatant and pellet fractions were analyzed by SDS-PAGE on 12% acrylamide gels. Protein bands were visualized with Coomassie Blue staining and analyzed by densitometry. For observation of cell structures, images were collected with a Zeiss Axioskop 2 Plus microscope using a Plan-Apochromat 100 × 1.4 numerical aperture objective lens and a Spot RT cooled charge-coupled device camera (Diagnostic Instruments, Sterling Heights, MI). Camera control and image enhancement were performed using MetaMorph Version 4.5 software (Universal Image Corp., Downingtown, PA). For the analysis of actin filament and mitochondrial morphology, ∼30 z-sections were obtained at 0.2-μm intervals through the entire cell. Out-of-focus light was removed by two-dimensional deconvolution using MetaMorph software, and further image processing, stacking of the series of images to a two-dimensional image was done with the NIH ImageJ. All cellular statistical analysis was based on cell counts of more than 100 for each sample. Mitochondria in living cells were visualized using a fusion protein in which GFP was fused to the mitochondrial signal sequence of citrate synthase as described previously (26.Bryan K.E. Wen K.K. Zhu M. Rendtorff N.D. Feldkamp M. Tranebjaerg L. Friderici K.H. Rubenstein P.A. J. Biol. Chem. 2006; 281: 20129-20139Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The construct used for expression of this protein was provided by Dr. Liza A. Pon, Columbia University, New York, NY (27.Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The actin cytoskeleton was visualized by fluorescence microscopy after staining fixed cells with rhodamine-phalloidin as described previously (19.McKane M. Wen K.K. Boldogh I.R. Ramcharan S. Pon L.A. Rubenstein P.A. J. Biol. Chem. 2005; 280: 36494-36501Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Vacuoles were observed following exposure of the cells to the dye FM 4–64 as described previously (28.Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1141) Google Scholar) and nuclear and mitochondrial DNAs were visualized following staining of the cells with 4′,6-diamidino-2-phenylindole as described previously (28.Vida T.A. Emr S.D. J. Cell Biol. 1995; 128: 779-792Crossref PubMed Scopus (1141) Google Scholar). Effects of Mutations in Vivo—To test for the effects of altering these hypothetical ionic interactions on actin function in vivo, we first assessed how the mutations in yeast actin altered growth characteristics of the cells under different growth conditions. Fig. 2A shows that all three of the mutant actins led to severely depressed extents of growth leveling off at ∼30% of the maximum level achieved by WT cells. Defects of this magnitude caused by actin mutations are often associated with mitochondria malfunction, because a functional cytoskeleton is required both for mitochondrial integrity and inheritance (27.Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 29.Yang H.C. Simon V. Swayne T.C. Pon L. Methods Cell Biol. 2001; 65: 333-351Crossref PubMed Google Scholar, 30.Boldogh I.R. Fehrenbacher K.L. Yang H.C. Pon L.A. Gene (Amst.). 2005; 354: 28-36Crossref PubMed Scopus (46) Google Scholar). To examine this possibility, we assessed the ability of the mutant cells to grow on glycerol as a sole carbon source. Yeast requires mitochondrial glycerol-3-phosphate dehydrogenase to ultimately convert the glycerol to dihydroxyacetone phosphate for use in glycolysis, and mitochondrial malfunction will interfere with this reaction (31.Klingenberg M. Eur. J. Biochem. 1970; 13: 247-252Crossref PubMed Scopus (208) Google Scholar, 32.Gancedo C. Gancedo J.M. Sols A. Eur. J. Biochem. 1968; 5: 165-172Crossref PubMed Scopus (195) Google Scholar). Fig. 2B shows that all three mutants, even the predicted hyperstable A167E mutant, fail to grow on glycerol as a fuel source. To gain insight into the nature of the mitochondrial malfunction, we transfected the cells with a plasmid carrying GFP to which was fused the citrate synthase mitochondrial targeting sequence (27.Fehrenbacher K.L. Yang H.C. Gay A.C. Huckaba T.M. Pon L.A. Curr. Biol. 2004; 14: 1996-2004Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Supplemental Fig. S1 shows that ∼90% of the GFP-expressing WT cells displayed normal tubular mitochondrial structures. Only ∼55–65% of the three mutant cells displayed GFP-fluorescent structures compared with ∼85% of WT cells, possibly due to mitochondrial structural defects. For the A167E and A167R cells, only about a third of the fluorescent cells displayed normal looking tubular mitochondrial structures. The rest of the cells either displayed aggregated structures or diffused fluorescence. Almost all of the fluorescent D275R cells contained brightly fluorescent dots instead of tubular structures indicative of severe mitochondrial disruption. Staining of the fixed cells with 4′,6-diamidino-2-phenylindole to visualize DNA showed that mitochondrial DNA was absent in virtually all of the Ala-167 mutants and showed aggregation in ∼75% of the D275R cells (supplemental Fig. S2). Nuclear segregation defects were seen in ∼10% of all three mutant cells. All three of the mutants exhibit varying degrees of temperature-sensitive growth on YPD medium (supplemental Fig. S3), and A167E also showed defective growth in hyperosmolar medium indicative of an impaired actin cytoskeleton (33.Chowdhury S. Smith K.W. Gustin M.C. J. Cell Biol. 1992; 118: 561-571Crossref PubMed Scopus (196) Google Scholar, 34.Slaninova I. Sestak S. Svoboda A. Farkas V. Arch. Microbiol. 2000; 173: 245-252Crossref PubMed Scopus (58) Google Scholar). Vacuole inheritance and morphology require a normal actin cytoskeleton (35.Bryant N.J. Stevens T.H. Microbiol. Mol. Biol. Rev. 1998; 62: 230-247Crossref PubMed Google Scholar, 36.Hill K.L. Catlett N.L. Weisman L.S. J. Cell Biol. 1996; 135: 1535-1549Crossref PubMed Scopus (185) Google Scholar), and the D275R and A167R cells displayed abnormal vacuole patterns (supplemental Fig. S4). Finally, abnormal actin cytoskeletal patterns were observed with rhodamine-phalloidin staining in all three mutants (supplemental Fig. S5). Effects of Mutations on G-actin Properties—Although we wished to use mutagenesis to examine the role of these hypothetical ionic bonds in stabilization of actin monomer-monomer interactions in the filament, it is possible that the mutations could have disruptive effects on the integrity of the monomer structure per se. We therefore assessed the effects of the mutations on G-actin thermostability and nucleotide exchange rates. We first used CD to measure the unfolding of actin as a function of temperature (Table 1). The A167E and A167R mutants exhibited apparent melting temperatures (Tm) within 1 degree of the WT Tm while the Tm of the D275R mutant was 8 °C lower than that of WT actin. However, it was stable within the temperature range in which the functional studies described below were performed. Because the nucleotide that is bound between subdomains 2 and 4 has a role in maintaining conformational integrity and stability of the monomer, we assessed the effects of the mutations on the rate of exchange of bound fluorescent ϵ-ATP from the actin in the presence of a vast excess of ATP. The A167E mutation caused a slower rate of exchange than WT actin, whereas the A167R and D275R mutations resulted in an increased rate of exchange (Table 1). However, the rates were within a factor of two of that of WT actin indicating that the overall effects of the mutations on actin structure were relatively small. Finally, we assessed the ability of the actin monomer binding protein cofilin to bind to WT and A167E, A167R, and D275R ATP-G-actins. Table 1 shows that the Kd of the cofilin for all of the ATP-G-actins is in the range of 1.2–2 μm, again suggesting no significant alteration of actin monomer conformation by the mutation.TABLE 1Effects of mutations on monomer thermostability, nucleotide exchange and binding to cofilin The number of experiments performed is indicated in parenthesis. Experiments were performed as described under “Experimental Procedures.”StrainThermostability apparent TmNucleotide exchange t½Kd of cofilin binding ATP actinC°sμmWT60.5 ± 0.5 (4)39 ± 4 (6)1.4 ± 0.8 (3)A167E61.2 ± 0.4 (3)50 ± 8 (5)1.1 ± 0.4 (3)A167R60 ± 2 (4)33 ± 4 (4)2.0 ± 0.4 (3)D275R52.6 ± 0.2 (3)30 ± 5 (6)1.18 ± 0.002 (2) Open table in a new tab Effects of Mutations on Actin Polymerization—Effects of the mutations on intermonomer ionic bonds in the filament might have been expected" @default.
• W2000224269 created "2016-06-24" @default.
• W2000224269 creator A5051679659 @default.
• W2000224269 creator A5052984331 @default.
• W2000224269 creator A5072032220 @default.
• W2000224269 date "2008-12-01" @default.
• W2000224269 modified "2023-09-29" @default.
• W2000224269 title "Role of Intermonomer Ionic Bridges in the Stabilization of the Actin Filament" @default.
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