Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus.

Sutoh, Kazuo; Ando, Mitsushige; Toyoshima, Yoko Y.

doi:10.1073/pnas.88.17.7711

Cited by 121 publications

(118 citation statements)

References 33 publications

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“…The reason for using this actin gene is that the N-terminal sequence of actin has been shown to be important for its function such as the interaction with myosin (29). Furthermore, the Tetrahymenaactin has only about 75%homologyto conventional actin and has been shown to display several unusual features (6) To see if the exogenous actin gene(s) has actually been introduced in the genome of these clones, the genomic DNAfrom each clone was analyzed by Southern hybridization using a fragment of the cloned genomic actin gene as a probe (Fig.…”

Section: Resultsmentioning

confidence: 99%

Recovery of Flagellar Inner-arm Dynein and the Fertilization Tubule in Chlamydomonas ida5 Mutant by Transformation with Actin Genes.

Ohara

Kato-Minoura

Kamiya

et al. 1998

Cell Struct. Funct.

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Key words: dynein mutant/flagellar actin/actin-like protein/chimeric actin gene/fertilization tubule ABSTRACT. The ida5 mutant of Chlamydomonas, first isolated as a mutant lacking a subset of axonemal inner-arm dyneins, has recently been shown to lack conventional actin owing to a serious mutation in its gene. It lacks inner-arm dyneins probably because actin is an essential subunit for their assembly. In addition, male gametes of ida5 are unable to produce the fertilization tubule, a structure that contains a core of actin filament bundles. To establish that those observed deficiencies are solely attributable to the loss of actin, and to provide a basis for future studies on the actin function in this organism, we examined in this study whether transformation of this mutant with cloned actin genes can rescue the mutant phenotypes. Cotransformation of the double mutant ida5arg2 with the wild-type actin gene and arginino-succinate lyase gene that suppresses the arg2 mutation yielded several transformants that displayed increased motility. All of them were found to have acquired the introduced actin gene in the genome and the product actin in the flagella, and regained the missing inner-arm dyneins and wild-type motility. In addition, most transformants also became able to grow the fertilization tubule whenmating reaction was induced. In addition to the wild-type actin gene, we also used a chimeric actin gene in which the N-terminal 12 amino-acid sequence of Chlamydomonasactin was replaced by that of the greatly divergent Tetrahymenaactin. Transformants with this gene also resulted in recovery of inner-arm dynein and 70-80% of the wild-type level of motility. These results established that the lack of inner-arm dynein and the fertilization tubule in ida5 are consequences of its loss of conventional actin. Furthermore, they demonstrate that Chlamydomonasoffers an excellent experimental system with which to study the structure-function relationship of actin by means of mutant analysis.

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Section: Resultsmentioning

confidence: 99%

Recovery of Flagellar Inner-arm Dynein and the Fertilization Tubule in Chlamydomonas ida5 Mutant by Transformation with Actin Genes.

Ohara

Kato-Minoura

Kamiya

et al. 1998

Cell Struct. Funct.

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“…In this study, the replacement of loop 2 with noninserted and B2-inserted sequences of human nonmuscle MHC-IIB added one and three more net negative charges compared with the wild type, respectively. The increase of the negative charges could be responsible for the reduction of the affinity for F-actin in Dic-B and Dic-B2 by repulsion of the N-terminal negative charges of actin (48,49).…”

Section: Discussionmentioning

confidence: 99%

Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms Using Dictyostelium Chimeric Myosin II

Takahashi

Hiratsuka

et al. 2001

Journal of Biological Chemistry

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The alternatively spliced isoform of nonmuscle myosin II heavy chain B (MHC-IIB) with an insert of 21 amino acids in the actin-binding surface loop (loop 2), MHC-IIB(B2), is expressed specifically in the central nervous system of vertebrates. To examine the role of the B2 insert in the motor activity of the myosin II molecule, we expressed chimeric myosin heavy chain molecules using the Dictyostelium myosin II heavy chain as the backbone. We replaced the Dictyostelium native loop 2 with either the noninserted form of loop 2 from human MHC-IIB or the B2-inserted form of loop 2 from human MHC-IIB(B2). The transformant Dictyostelium cells expressing only the B2-inserted chimeric myosin formed unusual fruiting bodies. We then assessed the function of chimeric proteins, using an in vitro motility assay and by measuring ATPase activities and binding to F-actin. We demonstrate that the insertion of the B2 sequence reduces the motor activity of Dictyostelium myosin II, with reduction of the maximal actin-activated ATPase activity and a decrease in the affinity for actin. In addition, we demonstrate that the native loop 2 sequence of Dictyostelium myosin II is required for the regulation of the actin-activated ATPase activity by phosphorylation of the regulatory light chain.

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“…Modification of the charge environment at the N terminus by either tag could affect these ionic interactions and interfere with the normal cross-bridge formation. Moreover, it has been shown that mutations in the N-terminal region of actin, which yielded a change of charge, affect myosin S1 binding to actin and thereby reduce in vitro motility (Aspenström and Karlsson, 1991;Aspenström et al, 1992;Sutoh et al, 1991;Miller et al, 1996). However, N-terminal mutations that do not induce charge changes may also affect actomyosin interactions.…”

Section: Tags At the N Terminus Of Actin May Influence Actin-myosin Imentioning

confidence: 99%

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

Brault¹,

Sauder

Reedy

et al. 1999

MBoC

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We have tested the impact of tags on the structure and function of indirect flight muscle (IFM)-specific Act88F actin by transforming mutant Drosophila melanogaster, which do not express endogenous actin in their IFMs, with tagged Act88F constructs. Epitope tagging is often the method of choice to monitor the fate of a protein when a specific antibody is not available. Studies addressing the functional significance of the closely related actin isoforms rely almost exclusively on tagged exogenous actin, because only few antibodies exist that can discriminate between isoforms. Thereby it is widely presumed that the tag does not significantly interfere with protein function. However, in most studies the tagged actin is expressed in a background of endogenous actin and, as a rule, represents only a minor fraction of the total actin. The Act88F gene encodes the only Drosophila actin isoform exclusively expressed in the highly ordered IFM. Null mutations in this gene do not affect viability, but phenotypic effects in transformants can be directly attributed to the transgene. Transgenic flies that express Act88F with either a 6x histidine tag or an 11-residue peptide derived from vesicular stomatitis virus G protein at the C terminus were flightless. Overall, the ultrastructure of the IFM resembled that of the Act88F null mutant, and only low amounts of C-terminally tagged actins were found. In contrast, expression of N-terminally tagged Act88F at amounts comparable with that of wild-type flies yielded fairly normal-looking myofibrils and partially reconstituted flight ability in the transformants. Our findings suggest that the N terminus of actin is less sensitive to modifications than the C terminus, because it can be tagged and still polymerize into functional thin filaments. INTRODUCTIONActins, a highly conserved family of cytoplasmic proteins, are among the most abundant proteins in eukaryotic cells. As a major component of the cytoskeleton, they control shape and motility in nonmuscle cells. In muscle, actin assembles into thin filaments, which together with interdigitating myosin thick filaments provide the framework for muscle contraction.Many organisms synthesize multiple isoforms of actin that are very similar in amino acid sequence even within the same cell. The differential expression of distinct actins as well as the high conservation of specific isoforms across species emphasize the functional importance of isoforms. In the case of actin, the question of how structure determines function appears to be particularly challenging. Considerable efforts have been made to understand how the different isoforms fulfill their various functions despite their extremely high sequence identity, and yet the basis of their functional diversity remains elusive. § Corresponding author. E-mail address: Schoenenberg@ ubaclu.unibas.ch. © 1999 by The American Society for Cell Biology 135Studying the specific role of a particular actin isoform has always been hampered by the difficulty of discriminating between the introduced a...

show abstract

Site-directed mutations of Dictyostelium actin: disruption of a negative charge cluster at the N terminus.

Cited by 121 publications

References 33 publications

Recovery of Flagellar Inner-arm Dynein and the Fertilization Tubule in Chlamydomonas ida5 Mutant by Transformation with Actin Genes.

Recovery of Flagellar Inner-arm Dynein and the Fertilization Tubule in Chlamydomonas ida5 Mutant by Transformation with Actin Genes.

Functional Characterization of Vertebrate Nonmuscle Myosin IIB Isoforms Using Dictyostelium Chimeric Myosin II

Differential Epitope Tagging of Actin in TransformedDrosophilaProduces Distinct Effects on Myofibril Assembly and Function of the Indirect Flight Muscle

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