Polypeptide subunits of dynein 1 from sea urchin sperm flagella

Bell, Christopher W.; Fronk, E.; Gibbons, I. R.

doi:10.1002/jss.400110305

Cited by 85 publications

(38 citation statements)

References 9 publications

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“…In addition, Egl also binds Dlc through a consensus light chain-binding site between amino acids 963 and 969 (Navarro et al 2004). BicD is able to recruit the dynein/dynactin complex (Hoogenraad et al 2003) and Dlc associates with other dynein subunits (Bell et al 1979). Thus, together with our evidence for Egl RNA binding through amino acids 1-814, it is now possible to build a working model of a complete link between minusend-directed mRNA signals and microtubules for the first time (Fig.…”

Section: Linkage Of Mrnas To the Motor Complex And The Regulation Of mentioning

confidence: 70%

Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Dienstbier¹,

Boehl²,

Li³

et al. 2009

Genes Dev.

186

318

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Cytoplasmic sorting of mRNAs by microtubule-based transport is widespread, yet very little is known at the molecular level about how specific transcripts are linked to motor complexes. In Drosophila, minus-end-directed transport of developmentally important transcripts by the dynein motor is mediated by seemingly divergent mRNA elements. Here we provide evidence that direct recognition of these mRNA localization signals is mediated by the Egalitarian (Egl) protein. Egl and the dynein cofactor Bicaudal-D (BicD) are the only proteins from embryonic extracts that are abundantly and specifically enriched on RNA localization signals from transcripts of gurken, hairy, K10, and the I factor retrotransposon. In vitro assays show that, despite lacking a canonical RNAbinding motif, Egl directly recognizes active localization elements. We also reveal a physical interaction between Egl and a conserved domain for cargo recruitment in BicD and present data suggesting that Egl participates selectively in BicD-mediated transport of mRNA in vivo. Our work leads to the first working model for a complete connection between minus-end-directed mRNA localization signals and microtubules and reveals molecular strategies that are likely to be of general relevance for cargo transport by dynein.[Keywords: Drosophila; dynein; mRNA localization; microtubule-based transport; stem-loop] Supplemental material is available at http://www.genesdev.org.

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Section: Linkage Of Mrnas To the Motor Complex And The Regulation Of mentioning

confidence: 70%

Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Dienstbier¹,

Boehl²,

Li³

et al. 2009

Genes Dev.

186

318

View full text Add to dashboard Cite

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“…However, we have no evidence to support this possibility. It is also worth noting that another high molecular weight MAP, dynein, the ATPase associated with ciliary and flagellar outer doublet microtubules, also contains LMW polypeptide components (32). Whether the MAP 1 complex is in some way related to dynein remains an interesting question.…”

Section: Resultsmentioning

confidence: 99%

Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1).

Vallee¹,

Davis²

1983

Proc. Natl. Acad. Sci. U.S.A.

100

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Microtubule-associated protein 1 (MAP 1; Mr, = 350,000) was analyzed by column chromatography of microtubule protein obtained from calf brain gray and'whitematter. Two low molecular weight proteins (LMW MAPs; Mr 28,000 'and 30,000) were found to cochromatograph with MAP 1 under all conditions examined. MAP 1 andthe LMW MAPs were purified from calf brain white matter as a complex containing approximately. equimolar amounts of the three species.. Urea (6 M) was used.to remove the LMW MAPs from. MAP 1.' Binding of MAP I to microtubules was unaffected by urea and occurred with or without the LMW species. Electron microscopy of microtubules composed of purified tubulin and either MAP 1 preparation revealed that, like MAP 2, MAP 1 has the appearance of a filamentous arm on the microtubule surface.

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“…In (2,25). Structural studies on isolated sea urchin LAD-i (26) and Chlamydomonas 18S dynein (27) have revealed that both dyneins have two globular heads connected by a Y-shaped stalk.…”

Section: Discussionmentioning

confidence: 99%

Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting.

King¹,

Otter²,

Witman³

1985

Proc. Natl. Acad. Sci. U.S.A.

110

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Monoclonal antibodies that recognize individual polypeptides of the outer arm dyneins of Chlamydomonas flagella were obtained and used to study the structural relationships between the various polypeptides. Immunoblot analysis showed that the y heavy chain of 12S dynein and the a and /3 heavy chains and M, 69 The dynein heavy chains migrate very close to one another in NaDodSO4/polyacrylamide gels and are well resolved only at very low protein loadings (2). Consequently, characterization of our antibodies by immunoblot analysis required (i) a reliable procedure for efficiently transferring high molecular weight polypeptides from NaDodSO4/polyacrylamide gels to nitrocellulose sheets without loss of resolution and (i) a sensitive method for detecting the transferred proteins on the nitrocellulose. The techniques that we have developed to fulfill these requirements were essential for unambiguous identification of the chains recognized by our antibodies and should be useful to a wide range of investigators needing to electrotransfer high molecular weight proteins or to detect nanogram amounts of protein on nitrocellulose. MATERIALS AND METHODSPreparation of Axonemes and Purification of Dynein. The isolation of flagella from Chlamydomonas reinhardtii (wildtype strain 1132D-) by the dibucaine method, preparation of axonemes, extraction with 0.6 M KCl, and subsequent purification of 12S and 18S dyneins by sucrose gradient centrifugation and hydroxylapatite column chromatography were as described (2, 4).Ram sperm tails were prepared from ejaculated sperm by modifications of methods described by Tash and Means (6) and Harrison (7). A pellet containing sperm tails and midpieces was treated with 0.05% Nonidet P-40 (BDH) in 30 mM Hepes, pH 7.5/5 mM MgSO4/1 mM dithiothreitol/0.5 mM EDTA/25 mM KCl/1 mM phenylmethylsulfonyl fluoride to remove the membranes. The demembranated axonemes were then collected by centrifugation at 30,500 x g and resuspended in electrophoresis sample buffer.Axonemes and latent-activity dynein-1 (LAD-1) from T. gratilla were the gift of

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Polypeptide subunits of dynein 1 from sea urchin sperm flagella

Cited by 85 publications

References 9 publications

Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Egalitarian is a selective RNA-binding protein linking mRNA localization signals to the dynein motor

Low molecular weight microtubule-associated proteins are light chains of microtubule-associated protein 1 (MAP 1).

Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting.

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