Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain.

We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors of the paralyzed central pair mutant pf6 (Luck, D.J.L., and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the beta-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an approximately 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by approximately 5 kb of genomic DNA (Mitchell, D.R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region that is predicted to encode a small alpha-helical coiled-coil domain of the beta-DHC. This domain may play a role in protein-protein interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.

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PF16 encodes a protein with armadillo repeats and localizes to a single microtubule of the central apparatus in Chlamydomonas flagella.

The Journal of Cell Biology ◽

10.1083/jcb.132.3.359 ◽

1996 ◽

Vol 132 (3) ◽

pp. 359-370 ◽

Cited By ~ 115

Author(s):

E F Smith ◽

P A Lefebvre

Keyword(s):

Protein Interactions ◽

Insertional Mutagenesis ◽

Flagellar Motility ◽

Wild Type ◽

Protein Protein Interactions ◽

Cellular Functions ◽

Central Pair ◽

Central Apparatus ◽

Immunofluorescence Labeling ◽

Armadillo Repeats

Several studies have indicated that the central pair of microtubules and their associated structures play a significant role in regulating flagellar motility. To begin a molecular analysis of these components we have generated central apparatus-defective mutants in Chlamydomonas reinhardtii using insertional mutagenesis. One paralyzed mutant recovered in our screen, D2, is an allele of a previously identified mutant, pf16. Mutant cells have paralyzed flagella, and the C1 microtubule of the central apparatus is missing in isolated axonemes. We have cloned the wild-type PF16 gene and confirmed its identity by rescuing pf16 mutants upon transformation. The rescued pf16 cells were wild-type in motility and in axonemal ultrastructure. A full-length cDNA clone for PF16 was obtained and sequenced. Database searches using the predicted 566 amino acid sequence of PF16 indicate that the protein contains eight contiguous armadillo repeats. A number of proteins with diverse cellular functions also contain armadillo repeats including pendulin, Rch1, importin, SRP-1, and armadillo. An antibody was raised against a fusion protein expressed from the cloned cDNA. Immunofluorescence labeling of wild-type flagella indicates that the PF16 protein is localized along the length of the flagella while immunogold labeling further localizes the PF16 protein to a single microtubule of the central pair. Based on the localization results and the presence of the armadillo repeats in this protein, we suggest that the PF16 gene product is involved in protein-protein interactions important for C1 central microtubule stability and flagellar motility.

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Domains in the 1α Dynein Heavy Chain Required for Inner Arm Assembly and Flagellar Motility in Chlamydomonas

The Journal of Cell Biology ◽

10.1083/jcb.146.4.801 ◽

1999 ◽

Vol 146 (4) ◽

pp. 801-818 ◽

Cited By ~ 57

Author(s):

Steven H. Myster ◽

Julie A. Knott ◽

Katrina M. Wysocki ◽

Eileen O'Toole ◽

Mary E. Porter

Keyword(s):

Heavy Chain ◽

Motor Domain ◽

Flagellar Motility ◽

Phosphate Binding ◽

Binding Motifs ◽

Phototactic Behavior ◽

Dynein Heavy Chain ◽

Dynein Motor ◽

Mutant Phenotypes

Flagellar motility is generated by the activity of multiple dynein motors, but the specific role of each dynein heavy chain (Dhc) is largely unknown, and the mechanism by which the different Dhcs are targeted to their unique locations is also poorly understood. We report here the complete nucleotide sequence of the Chlamydomonas Dhc1 gene and the corresponding deduced amino acid sequence of the 1α Dhc of the I1 inner dynein arm. The 1α Dhc is similar to other axonemal Dhcs, but two additional phosphate binding motifs (P-loops) have been identified in the NH2- and COOH-terminal regions. Because mutations in Dhc1 result in motility defects and loss of the I1 inner arm, a series of Dhc1 transgenes were used to rescue the mutant phenotypes. Motile cotransformants that express either full-length or truncated 1α Dhcs were recovered. The truncated 1α Dhc fragments lacked the dynein motor domain, but still assembled with the 1β Dhc and other I1 subunits into partially functional complexes at the correct axoneme location. Analysis of the transformants has identified the site of the 1α motor domain in the I1 structure and further revealed the role of the 1α Dhc in flagellar motility and phototactic behavior.

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Insights into the Structural Organization of the I1 Inner Arm Dynein from a Domain Analysis of the 1β Dynein Heavy Chain

Molecular Biology of the Cell ◽

10.1091/mbc.11.7.2297 ◽

2000 ◽

Vol 11 (7) ◽

pp. 2297-2313 ◽

Cited By ~ 69

Author(s):

Catherine A. Perrone ◽

Steven H. Myster ◽

Raqual Bower ◽

Eileen T. O'Toole ◽

Mary E. Porter

Keyword(s):

Heavy Chain ◽

Structural Organization ◽

Microscopic Analysis ◽

Transcription Unit ◽

Light Chains ◽

Flagellar Motility ◽

Electron Microscopic ◽

Dynein Heavy Chain ◽

Complex Transformation ◽

Motility Mutant

To identify domains in the dynein heavy chain (Dhc) required for the assembly of an inner arm dynein, we characterized a new motility mutant (ida2-6) obtained by insertional mutagenesis.ida2-6 axonemes lack the polypeptides associated with the I1 inner arm complex. Recovery of genomic DNA flanking the mutation indicates that the defects are caused by plasmid insertion into theDhc10 transcription unit, which encodes the 1β Dhc of the I1 complex. Transformation with Dhc10 constructs encoding <20% of the Dhc can partially rescue the motility defects by reassembly of an I1 complex containing an N-terminal 1β Dhc fragment and a full-length 1α Dhc. Electron microscopic analysis reveals the location of the missing 1β Dhc motor domain within the axoneme structure. These observations, together with recent studies on the 1α Dhc, identify a Dhc domain required for complex assembly and further demonstrate that the intermediate and light chains are associated with the stem regions of the Dhcs in a distinct structural location. The positioning of these subunits within the I1 structure has significant implications for the pathways that target the assembly of the I1 complex into the axoneme and modify the activity of the I1 dynein during flagellar motility.

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Isolation of dynein heavy chain cDNAs from trout testis which predict an extensive carboxyl-terminal alpha-helical coiled-coil domain.

The EMBO Journal ◽

10.1002/j.1460-2075.1989.tb03565.x ◽

1989 ◽

Vol 8 (6) ◽

pp. 1727-1734 ◽

Cited By ~ 14

Author(s):

A. T. Garber ◽

J. D. Retief ◽

G. H. Dixon

Keyword(s):

Heavy Chain ◽

Coiled Coil ◽

Dynein Heavy Chain ◽

Coiled Coil Domain ◽

Carboxyl Terminal

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Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein

Journal of Cell Science ◽

10.1242/jcs.107.3.497 ◽

1994 ◽

Vol 107 (3) ◽

pp. 497-506 ◽

Cited By ~ 14

Author(s):

C.G. Wilkerson ◽

S.M. King ◽

G.B. Witman

Keyword(s):

Heavy Chain ◽

Coiled Coil ◽

Cytoplasmic Dynein ◽

Complete Sequence ◽

Dynein Heavy Chain ◽

Heavy Chains ◽

Microtubule Binding Domain ◽

Helical Content ◽

Axonemal Dynein ◽

Dynein Heavy Chains

We report here the complete sequence of the gamma dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The gamma dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high alpha-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the gamma dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.

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Coiled-coil networking shapes cell molecular machinery

Molecular Biology of the Cell ◽

10.1091/mbc.e12-05-0396 ◽

2012 ◽

Vol 23 (19) ◽

pp. 3911-3922 ◽

Cited By ~ 44

Author(s):

Yongqiang Wang ◽

Xinlei Zhang ◽

Hong Zhang ◽

Yi Lu ◽

Haolong Huang ◽

...

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Coiled Coil ◽

Coiled Coils ◽

Protein Protein Interactions ◽

Full Spectrum ◽

Kinetochore Assembly ◽

Genome Wide ◽

Molecular Machinery ◽

Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

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Lamellipodial localization ofDictyosteliummyosin heavy chain kinase A is mediated via F-actin binding by the coiled-coil domain

FEBS Letters ◽

10.1016/s0014-5793(02)02494-8 ◽

2002 ◽

Vol 516 (1-3) ◽

pp. 58-62 ◽

Cited By ~ 18

Author(s):

Paul A Steimle ◽

Lucila Licate ◽

Graham P Côté ◽

Thomas T Egelhoff

Keyword(s):

Heavy Chain ◽

Coiled Coil ◽

Actin Binding ◽

Coiled Coil Domain ◽

Kinase A

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Yeast Surface Two-hybrid for Quantitative in Vivo Detection of Protein-Protein Interactions via the Secretory Pathway

Journal of Biological Chemistry ◽

10.1074/jbc.m109.001743 ◽

2009 ◽

Vol 284 (24) ◽

pp. 16369-16376 ◽

Cited By ~ 23

Author(s):

Xuebo Hu ◽

Sungkwon Kang ◽

Xiaoyue Chen ◽

Charles B. Shoemaker ◽

Moonsoo M. Jin

Keyword(s):

Protein Interactions ◽

Secretory Pathway ◽

Coiled Coil ◽

Affinity Maturation ◽

Antibody Binding ◽

Soluble Form ◽

Protein Protein Interactions ◽

Yeast Surface ◽

Two Hybrid

A quantitative in vivo method for detecting protein-protein interactions will enhance our understanding of protein interaction networks and facilitate affinity maturation as well as designing new interaction pairs. We have developed a novel platform, dubbed “yeast surface two-hybrid (YS2H),” to enable a quantitative measurement of pairwise protein interactions via the secretory pathway by expressing one protein (bait) anchored to the cell wall and the other (prey) in soluble form. In YS2H, the prey is released either outside of the cells or remains on the cell surface by virtue of its binding to the bait. The strength of their interaction is measured by antibody binding to the epitope tag appended to the prey or direct readout of split green fluorescence protein (GFP) complementation. When two α-helices forming coiled coils were expressed as a pair of prey and bait, the amount of the prey in complex with the bait progressively decreased as the affinity changes from 100 pm to 10 μm. With GFP complementation assay, we were able to discriminate a 6-log difference in binding affinities in the range of 100 pm to 100 μm. The affinity estimated from the level of antibody binding to fusion tags was in good agreement with that measured in solution using a surface plasmon resonance technique. In contrast, the level of GFP complementation linearly increased with the on-rate of coiled coil interactions, likely because of the irreversible nature of GFP reconstitution. Furthermore, we demonstrate the use of YS2H in exploring the nature of antigen recognition by antibodies and activation allostery in integrins and in isolating heavy chain-only antibodies against botulinum neurotoxin.

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The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.

Molecular Biology of the Cell ◽

10.1091/mbc.8.4.607 ◽

1997 ◽

Vol 8 (4) ◽

pp. 607-620 ◽

Cited By ~ 83

Author(s):

S H Myster ◽

J A Knott ◽

E O'Toole ◽

M E Porter

Keyword(s):

Heavy Chain ◽

Insertional Mutagenesis ◽

Northern Blot Analysis ◽

Immunoblot Analysis ◽

Transcription Unit ◽

Peptide Sequence ◽

Dynein Heavy Chain ◽

The Individual ◽

Intermediate Chains ◽

Specific Peptide

Multiple members of the dynein heavy chain (Dhc) gene family have been recovered in several organisms, but the relationships between these sequences and the Dhc isoforms that they encode are largely unknown. To identify Dhc loci and determine the specific functions of the individual Dhc isoforms, we have screened a collection of motility mutants generated by insertional mutagenesis in Chlamydomonas. In this report, we characterize one strain, pf9-3, in which the insertion event was accompanied by a deletion of approximately 13 kb of genomic DNA within the transcription unit of the Dhc1 gene. Northern blot analysis confirms that pf9-3 is a null mutation. Biochemical and structural studies of isolated axonemes demonstrate that the pf9-3 mutant fails to assemble the I1 inner arm complex, a two-headed dynein isoform composed of two Dhcs (1 alpha and 1 beta) and three intermediate chains. To determine if the Dhc1 gene product corresponds to one of the Dhcs of the I1 complex, antibodies were generated against a Dhc1-specific peptide sequence. Immunoblot analysis reveals that the Dhc1 gene encodes the 1 alpha Dhc subunit. These studies thus, identify the first inner arm Dhc locus to be described in any organism and further demonstrate that the 1 alpha Dhc subunit plays an essential role in the assembly of the I1 inner arm complex.

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