Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat shock protein 40 in sperm flagella of the ascidian Ciona intestinalis

Abstract

Members of the heat-shock protein (HSP)40 regulate the protein folding activity of HSP70 proteins and help the functional specialization of this molecular chaperone system in various types of cellular events. We have recently identified Hsp40 as a component of flagellar axoneme in the ascidian Ciona intestinalis, suggesting a correlation between Hsp40 related chaperone system and flagellar function. In this study, we have found that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution and comigrates with radial spoke protein (RSP)3 along with several proteins as a complex through gel filtration and ion exchange columns. Peptide mass fingerprinting with matrix-assisted laser desorption ionization/time of flight/mass spectrometry revealed that other proteins in the complex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally unknown 33-kDa protein. A spoke head protein, LRR37, is not included in the complex, suggesting that the complex constructs the stalk of radial spoke. Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk, possibly at the junction between spoke head and the stalk.

Figure 1.

Phylogenic tree of the members of Hsp40 family in human, mouse, and Ciona. Note that CiAxHsp40 (asterisk) belongs to DjB1/B4 class (shaded). Two Hsp70s, mouse Hsp70-2 (accession no. I49761) and mouse Hsp70t (accession no. BC004714) were used as outgroups. Ciona Hsp40s were searched at http://genome.jgi-psf.org/Ciona4/Ciona4.home.html. The names of the gene number are indicated. The number on the branch represents the percentage of times that a node was supported in 1000 bootstrap pseudoreplications. The accession numbers of human mouse Hsp40s are hsDjA1, D13388; hsDjA2, AJ001309; hsDjA3, AF061749; hsDjB1, X62421; hsDjB2, X63368; hsDjB4, U40992; hsDjB5, AF088982; hsDjB6, AB014888; hsDjB8, BC029521; hsDjB9, AF083247; hsDjB11, AJ250137; hsDjC1, AK027263; hsDjC2, X98260; hsDjC3, U28424; hsDjC4, AF036874; hsDjC5, AL118506; hsDjC7, U46571; hsDjC8, AF083190; mmDjA1, AF055664; mmDjA2, AB028853; mmDjA3, AY009320; mmDjB1, AB028272; mmDjB3, U95607; mmDjB4, AK008537; mmDjB5, AF092536; mmDjB6, AF035962; mmDjB7, AB028855; mmDjB8, AB028856; mmDjB9, AB028857; mmDjB1, 0AB028858; mmDjB1, 1BC003999; mmDjB1, 2AB028860; mmDjC1, L16953; mmDjC2, U53208; mmDjC3, U28423; mmDjC4, AF036875; mmDjC5, U39320; and mmDjC7, BC023681.

Figure 2.

Expression patterns of several members of Hsp40 in adult tissues of C. intestinalis. Data are based on >480,000 EST data collected from several Ciona tissues (http://ghost.zool.kyoto-u.ac.jp/indexr1.html). The vertical values in the histogram reflect the occurrence of the gene (number of occurrence/number of total ESTs collected). The DjB1/B4 class of Hsp40 is highlighted. BC, blood cells; E, endostyle; G, gonad; H, heart; NC, neural complex; T, testis.

Figure 4.

Western blot of several fractions from chemical dissection of sperm axonemes. (A) Sperm flagella (Fla) were fractionated into Triton X-100 extract (TX), 0.6 M KCl extract of the axonemes (KCl), the supernatant (TD), and the pellet (P) after Tris-EDTA treatment of the KCl-extracted axonemes. (B) Sperm flagella (Fla) were fractionated into Triton X-100 extract (TX), 0.6 M KCl extract of the axonemes (KCl), the supernatant (KI), and the pellet (P) after 0.5 M KI treatment of the KCl-extracted axonemes. Both data show the Coomassie-stained protein pattern (left) and corresponding Western blot with anti-CiAxHsp40 (right). The arrows show the position of CiAxHsp40.

Figure 3.

Immunofluorescence microscopy showing the localization of CiAxHsp40 along sperm flagella. Ciona sperm were fixed, stained with anti-CiAxHsp40 antibody (red), and counterstained with DAPI (blue). Differential interference contrast and merged fluorescent images also are shown. Normal mouse serum was used in the control experiment. Anti-CiAxHsp40 antibody clearly stained the flagellum, although slight nonspecific staining on the head was observed, as also with control serum. Bar, 10 μm.

Figure 5.

Immunoelectron microscopy showing the localization of CiAxHsp40 in the Ciona axonemes after 0.6 M KCl treatment. Images a-e show immunogold labeling with anti-CiAxHsp40 antibody. Image f shows control (secondary antibody alone). The gold particles are observed at the distal part of the radial spokes. Bar, 100 nm.

Figure 6.

Poros HQ ion exchange column chromatography of the KI extract from KCl-treated axonemes. Proteins were eluted by a programmed NaCl gradient as shown by dotted line. The bottom blot shows the elution profile of CiRSP3, obtained by Western blotting with anti-CiRSP3 antibody.

Figure 7.

Elution profiles of CiRSP3 and CiAxHsp40 through Superose 6 gel filtration. Top, protein pattern in each fraction, visualized by silver staining. Bottom, elution patterns of CiRSP3 (49 kDa) and CiAxHsp40 (37 kDa), examined by Western blotting.

Figure 8.

Protein composition of RSP3-containing fraction obtained after Superose 6 gel filtration. The gel was visualized by Coomassie Brilliant Blue. Proteins subjected to peptide mass fingerprinting are shown by arrows with their molecular masses on the right. The asterisk shows the 53- and 52-kDa bands of α- and β-tubulins.

Figure 9.

Immunoprecipitation of CiRSP3-containing subcomplex in the KI extract of KCl-treated axonemes. The Coomassie-stained blot on the left shows the protein composition of the precipitate, with the major components indicated by arrowheads. Asterisks show the position of tubulins. Other four blots show the immunoblots of the precipitate with antibodies against CiMORN40, CiAxHsp40, CiRSp33, and LRR37.