Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the Ascidian Ciona intestinalis

Abstract

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidian Ciona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported in Chlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.

Figure 1

Two-dimensional gel electrophoresis of flagellar axonemes of C. intestinalis. Silver protein staining (left) and corresponding immunoblot with anti-axoneme antiserum (right) are shown. Axonemal proteins were first separated by isoelectric focusing (pH 3–10), followed by SDS-PAGE with 7% (A) and 12% (B) polyacrylamide for separating gels. Marker positions for isoelectric points or molecular mass (kilodaltons) are indicated.

Figure 2

(A) Nucleotide and deduced amino acid sequences of the full-length cDNA that encodes the leucine-rich repeat protein LRR 37 from C. intestinalis. These sequence data are available from GenBank/DDBJ/EMBL under the accession number AB079056. The regions of LRR are underlined. (B) Multiple alignment of Ciona LRR37, mouse B7, and human B7 proteins by CLUSTALW. Asterisks, colons, and dots indicate identical residues in all sequences in the alignment, conserved substitutions, and semiconserved substitutions, respectively.

Figure 3

Two-dimensional gel electrophoresis of flagellar axonemes of C. intestinalis. Left, Coomassie protein staining and right, corresponding immunoblot with anti-LRR37 antibody. Axonemal proteins were first separated by isoelectric focusing (pH 3–10), followed by SDS-PAGE with 10% polyacrylamide for separating gel. Marker positions for isoelectric points or molecular mass (kilodaltons) are indicated. The antibody recognized a single protein spot with molecular mass or pI of 40 kDa or 4.65, respectively.

Figure 4

Immunoblot of several fractions of axonemes by anti-LRR37 antibody. Flagella (Fla) were demembranated with Triton X-100 (TX) to obtain the axonemes. Axonemes were then successively treated with 0.6 M KCl solution (KCl), followed by the dialysis against a low ionic strength (TD, supernatant; and P, pellet after ultracentrifugation). A 40-kDa immunoreactive band was exclusively detected in TD supernatant.

Figure 5

Immunofluorescence microscopy with anti-LRR37 antibody. Left, differential interference contrast image of Ciona sperm. Right, corresponding immunostaining with Alexa 546-conjugated secondary antibody. Bar, 10 μm.

Figure 6

Immunogold electron microscopy with anti-LRR37 antibody. Labeling patterns in cross sections of intact (a and b) and partially disrupted axonemes (c–g), longitudinal sections of partially disrupted axonemes (h–k) are shown. Bar, 200 nm.

Figure 7

(A) Two-dimensional gel electrophoresis of flagellar axonemes of C. intestinalis. Coomassie protein staining pattern (right) and corresponding immunoblot with anti-RSP3 antibody (right) are shown. Axonemal proteins were first separated by isoelectric focusing (pH 4–7), followed by SDS-PAGE with 7% polyacrylamide for separating gel. Marker positions for molecular mass (kilodaltons) and pI are indicated. The antibody recognized a single protein spot with molecular mass or pI of 49 kDa or 5.07, respectively. (B) Immunoblot of several fractions of axonemes by anti-RSP3 antibody. Flagella (Fla) were demembranated with Triton X-100 (TX) to obtain the axonemes. Axonemes were then successively treated with 0.6 M KCl solution (KCl), followed by the dialysis against a low ionic strength (TD, supernatant; and P, pellet after ultracentrifugation). A 49-kDa immunoreactive band was mostly detected in TD pellet.

Figure 8

Immunogold localization of RSP3. Labeling patterns in cross sections of the axoneme retaining 9 + 2 structure (a and b) or in partially disrupted axonemes (c–g) are shown. Bar, 200 nm.

Figure 9

Gel filtration of KI extract by BioSilect SEC400 column. (A) KI extract from KCl-treated axonemes was loaded on a gel filtration column (300 × 7.8 mm) and separated at a flow rate of 1.0 ml/min. Fractions (200 μl) were collected. The numbers above the chromatogram indicate the fraction number. Arrows show the elution positions of molecular mass markers as follows: thyroglobulin (670 kDa), immunoglobulin G (150 kDa), ovalbumin (44 kDa), and myoglobin (17 kDa). (B) Proteins in the fraction 4–25 were separated by 10% SDS-PAGE and immunoblotted by anti-LRR37 and anti-RSP3 antibodies. Both proteins were coeluted at around the position with molecular mass of 1300 kDa.