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. 2004 Dec;15(12):5431-42.
doi: 10.1091/mbc.e04-08-0694. Epub 2004 Oct 6.

IC138 Is a WD-repeat Dynein Intermediate Chain Required for Light Chain Assembly and Regulation of Flagellar Bending

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Free PMC article

IC138 Is a WD-repeat Dynein Intermediate Chain Required for Light Chain Assembly and Regulation of Flagellar Bending

Triscia W Hendrickson et al. Mol Biol Cell. .
Free PMC article

Abstract

Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform.

Figures

Figure 1.
Figure 1.
Inner arm dynein I1 and IC138 cloning strategy. (A) Illustration of the inner arm dynein I1. The I1 dynein complex is composed of two heavy chains (1α Dhc and 1β Dhc), three intermediate chains (IC140, IC138, and IC97—also called IC110), and several light chains (LC8, LC7a, LC7b, Tctex1, and Tctex2b). (B) Primers (P5S and P1AS) were designed from sequence obtained from purified IC138 and used for RT-PCR. Sequences obtained by RT-PCR were then used to design additional primers for screening Chlamydomonas genomic libraries. The sequences obtained from those screens were then used to screen cDNA libraries to generate the full-length IC138 cDNA. The IC138 gene contains 11 exons (solid bars). The IC138 sequence can be obtained from GenBank under the accession no. AY743342.
Figure 2.
Figure 2.
The IC138 gene encodes a WD-repeat containing protein. The IC138 cDNA predicts a protein of 1057 amino acids and ∼111 kDa with a calculated pI of 5.73. Peptide sequences obtained from microsequencing purified IC138 are underlined; the N-terminal sequence that was used to create the His-tagged fusion protein for the purpose of producing the IC138 polyclonal antibody is italicized. IC138, like other IC dyneins, is predicted to contain seven WD-repeats, which are indicated in bold. The residues enclosed in the shaded box are missing in bop5-1.
Figure 3.
Figure 3.
IC138 structural domains and homologues. (A) Schematic illustration of the dynein intermediate chains comparing the positions of the seven WD/β-sheet repeats among several axonemal dynein intermediate chains. Alignment of these sequences indicates that the WD/β-sheet repeats are all clustered within the C-terminal portion of the proteins. (B) The MegAlign Program (DNASTAR) was used to generate a phylogenetic tree of dynein intermediate chains. (C) Alignment of the Chlamydomonas IC138 (C.r. IC138) and its homologues human IC138 (DNAI3), mouse IC138 (Dnai3).
Figure 3.
Figure 3.
IC138 structural domains and homologues. (A) Schematic illustration of the dynein intermediate chains comparing the positions of the seven WD/β-sheet repeats among several axonemal dynein intermediate chains. Alignment of these sequences indicates that the WD/β-sheet repeats are all clustered within the C-terminal portion of the proteins. (B) The MegAlign Program (DNASTAR) was used to generate a phylogenetic tree of dynein intermediate chains. (C) Alignment of the Chlamydomonas IC138 (C.r. IC138) and its homologues human IC138 (DNAI3), mouse IC138 (Dnai3).
Figure 3.
Figure 3.
IC138 structural domains and homologues. (A) Schematic illustration of the dynein intermediate chains comparing the positions of the seven WD/β-sheet repeats among several axonemal dynein intermediate chains. Alignment of these sequences indicates that the WD/β-sheet repeats are all clustered within the C-terminal portion of the proteins. (B) The MegAlign Program (DNASTAR) was used to generate a phylogenetic tree of dynein intermediate chains. (C) Alignment of the Chlamydomonas IC138 (C.r. IC138) and its homologues human IC138 (DNAI3), mouse IC138 (Dnai3).
Figure 4.
Figure 4.
IC138 is truncated in bop5-1. (A) Abbreviated map of Chlamydomonas linkage group XII/XIII. The IC138 gene was mapped to the left arm of linkage group XII/XIII based on linkage to the genetic marker pf9 (∼29 cM) and a series of molecular markers as described in Materials and Methods. (B) Sequence analysis of IC138 from the bop5-1 allele revealed a point mutation at nucleotide 4725 that results in a premature stop codon, 974E → STOP. (C) Positions of the seven WD/β-sheet repeats are indicated by boxes. The bop5-1 IC138 nucleotide sequence predicts a truncated protein that lacks the last 84 amino acids, including the last WD-repeat (arrow), producing a truncated protein. (D) Axonemes from wild-type and bop5-1 cells probed with a polyclonal antibody generated against a fusion protein containing the first N-terminal 148 amino acids of IC138 (Figure 2, italicized sequence), which specifically recognizes a protein at ∼118 kDa corresponding to IC138, and a polyclonal IC140 antibody (Yang and Sale, 1998).
Figure 5.
Figure 5.
IC138 rescues motility in bop5-1. bop5-1 cells that had been transformed with full-length IC138 and stable diploids containing both the wild-type IC138 gene and the bop5-1 IC138 gene were analyzed. (A) Swimming speeds of freely swimming cells were measured. The decreased swimming speed seen in bop5-1 is rescued by wild-type IC138. (B) By Western analysis, only the full-length IC138 is found in the axonemes of the stable diploids (#1, #2, and #4) and the bop5-1 cells transformed with the wild-type IC138 gene (2A, E6).
Figure 6.
Figure 6.
LC7b fails to assemble in I1 from bop5-1. (A) Western analyses were performed on axonemes isolated from wild-type and bop5-1 cells by using antibodies to the 1α dynein heavy chain (1α Dhc), two intermediate chains (IC138 and IC140), and the light chains (LC8, Tctex1, and Tctex2b). (B) Silver-stained SDS-PAGE of I1 fractions indicating that IC97 (arrowhead) is assembled in axonemes from bop5-1 cells in an amount comparable with the wild-type positive control derived from pf28 axonemes and lacking in the negative control derived from pf28pf30 axonemes. IC140, IC138, and truncated IC138 are indicated with asterisks. (C) Further analysis of bop5-1 and the double mutant bop5-1oda7 reveals that LC7b, but not LC7a fails to assemble when IC138 is truncated. The tubulin region of the gel was excised and Coomassie stained to demonstrate protein loads.
Figure 7.
Figure 7.
EDC cross-links IC138 to an ∼97-kDa axonemal protein. Axonemal proteins were isolated from wild-type (WT), bop5-1 and 5A (truncated IC140) cells and cross-linked using the zero-length cross-linker EDC. The samples were then separated on a 3–6% gradient gel and transferred to nitrocellulose. The blots were probed with α-IC140 or α-IC138. The IC138 and IC140 EDC cross-linked products (indicated by the asterisk) are coincidentally at the same position for WT axonemes (∼250 kDa) but has shifted in the bop5-1 IC138 blot as well as the 5A IC140 blot by ∼97 kDa in each case, indicating that IC138 and IC140 both interact with an axonemal protein of ∼97 kDa.
Figure 8.
Figure 8.
IC138 is hyperphosphorylated in radial spoke and central pair mutants. To determine whether of the phosphorylation state of IC138 is altered in paralyzed flagellar mutants, axonemes isolated from wild-type, pf17, pf18, mia2-1, bop5-1, and bop5-1pf17 were treated with CIP. (A) Based on a comparison of CIP-treated (-) versus control, untreated (+) axonemes, IC138 seems to be hyperphosphorylated in mia2-1 and the paralyzed mutants pf17, pf18, and bop5-1pf17. In contrast, IC138 from wild type and bop5-1 is not hyperphosphorylated. (B) Flagella were isolated and demembranated immediately preceding the addition of ATP and protease. Microtubule sliding velocity is expressed as micrometers per second.

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