Cloning of C. reinhardtii IFT46
The gene and cDNA encoding C. reinhardtii IFT46 were cloned as described in Materials and methods. The cDNA (accession no. DQ787426) contains a 1,035-nt ORF predicted to encode a 37.9-kD protein () with a pI of 4.61. The cDNA has a stop codon 18 nt upstream of the predicted start codon, and a polyA consensus sequence at nt 1,703–1,707. ESTs have a polyA tail 12–14 nt downstream of the polyA consensus sequence. Therefore, the ORF is complete. No structural domains or motifs were identified within the sequence.
Figure 1. Identification and characterization of C. reinhardtii IFT46. (A) Predicted amino acid sequence of IFT46 and alignment with orthologues from other organisms. CrIFT46 is highly conserved from aa 100–315. Amino acid identities are marked with asterisks; (more ...)
IFT46 was initially identified as an IFT complex B protein, based on its cosedimentation with other complex B proteins in sucrose gradients (Cole et al., 1998
). Characterization of our cloned protein indicates that it behaves exactly as expected of a 46-kD complex B protein, as follows: a) six unique peptides corresponding to the cloned protein were identified in the membrane plus matrix fraction of the flagellar proteome, but no peptides from it were found in any other fraction, which is a distribution typical for IFT particle proteins but unusual for non-IFT proteins (Pazour et al., 2005
); b) using real-time PCR, we found that expression of the protein is up-regulated 10.4 ± 2.03-fold (SEM) upon deflagellation, which is characteristic of flagellar proteins, including other complex B proteins (Pazour et al., 2005
); c) an antibody to a peptide contained in the cloned protein was generated and shown to react specifically with a single protein of Mr
~46,000 in Western blots of whole cell lysates (); d) immunofluorescence microscopy with the antibody as probe showed that the majority of the cloned protein is located in the basal body region, with a lesser amount in puncta along the length of the flagella (), is a distribution identical to that of other IFT particle proteins (Cole et al., 1998
; Deane et al., 2001
); moreover, the protein colocalized with complex B protein IFT172, but not complex A protein IFT139 (see the section Complex A and B proteins are located in distinct compartments in the basal body region; ); and e) when the flagellar membrane plus matrix fraction was analyzed by sucrose density gradient centrifugation, the cloned protein co-migrated precisely with IFT81, a complex B protein, but not with IFT139, a complex A protein (). These results indicate that the cloned protein is indeed IFT46, and confirm that IFT46 is a complex B protein.
Figure 6. Complex A and B proteins differ in cellular distribution, and loss of IFT46 affects the cell body localization of IFT172, but not of IFT139. (A) ift46 cells were double labeled either with antibodies to IFT172 (a) and tubulin (b) or with antibodies to (more ...)
IFT46 is highly conserved
Database searches revealed that IFT46 is conserved across organisms that have cilia, including Danio rerio
(XP_694278; BLAST E, 1e-47), Apis melifera
(XP_396519; BLAST E, 1e-47), Drosophila melanogaster
(NP_609890; BLAST E, 1e-17), Mus musculus
(NP_076320; BLAST E, 2e-61), and Homo sapiens
(CAB66868; BLAST E, 8e-62). The protein is also homologous to C. elegans
DYF-6 (NP_741887; BLAST E, 9e-34), which was recently reported to undergo IFT in dendritic cilia when fused to GFP (Bell et al., 2006
). No similar sequence was found in nonciliated organisms, including Saccharomyces cerevisiae
and Arabidopsis thaliana
The middle portions of the C. reinhardtii and mammalian proteins are highly similar (51% identity, 72% similarity; ), making it likely that they are orthologous proteins. To test this, the putative mouse orthologue was Flag tagged and expressed in IMCD3 cells, a mouse kidney cell line. Immunofluorescence microscopy revealed that the Flag-tagged protein localized specifically to primary cilia (). To determine if the mammalian protein was part of IFT complex B, lysates were prepared from IMCD3 cells expressing either IFT46-Flag, IFT20-Flag (positive control), or GFP-Flag (negative control) and immunoprecipitated with an anti-Flag antibody. In each case, the Flag-tagged proteins were highly enriched in the immunoprecipitates (, top). Western blots showed that complex B proteins IFT88 and IFT57, but not complex A protein IFT140 or a control protein, were coimmunoprecipitated from lysates of the IFT46-Flag–expressing cells (, bottom). Similarly, in the positive control, IFT88 and IFT57, but not IFT140, were coimmunoprecipitated. No IFT proteins were coimmunoprecipitated from the lysate of cells expressing GFP-Flag. Therefore, mammalian IFT46 localizes to cilia and is tightly associated with complex B proteins, but not with complex A proteins.
Figure 2. The mouse orthologue of CrIFT46 is also an IFT complex B protein. (A) MmIFT46 localizes in cilia. IMCD3 cells expressing Flag-tagged MmIFT46 were labeled with antibodies to Flag (red), IFT20 (green), which localizes to Golgi in addition to cilia, and (more ...)
IFT46 is necessary for flagellar assembly
To investigate the role of IFT46 in intraflagellar transport, we screened a collection of C. reinhardtii insertional mutants by Southern blotting and identified one strain, T8a44-11, with a defect in the IFT46 gene. Analysis by PCR showed that the mutant allele, which we term ift46-1, has a deletion or disruption somewhere between the fourth and the seventh exon of the IFT46 gene (). This mutant has short, stumpy flagella.
Figure 3. The nonmotile phenotype of YH6 is caused by a null mutation in the IFT46 gene. (A) IFT46 gene structure. There are two PstI sites (P) within the IFT46 gene. Three primer sets used for PCR to map the mutation region in the mutant are indicated by arrowheads. (more ...)
Strain T8a44-11 was backcrossed to wild-type cells, and a progeny, YH6, which lacked the pf1 mutation carried by the parental strain, was selected for detailed characterization. As in T8a44-11, the IFT46 gene in YH6 is disrupted, as shown by Southern blotting (, lane 2). No IFT46 can be detected in lysates of YH6 cells (, lane 2), indicating that IFT46 is not expressed in YH6 cells. Thus, the mutant allele is a null allele (see also the section The 3′ end of the IFT46 gene is transcribed in Supift461 cells). Like T8a44-11, YH6 cells have short, stumpy flagella that barely extend beyond the flagellar collar (). The flagella are nonmotile.
Figure 4. Flagellar assembly is defective in ift46 mutant cells. (A) Electron micrographs show that ift46 flagella are short with normal basal bodies and transition zones. The mutant flagella lack dynein arms, and central pair assembly is defective. (a–c) (more ...)
To confirm that the mutant phenotype of YH6 was caused by the disruption of the IFT46 gene, YH6 cells were transformed with a 4.8-kb fragment containing only the wild-type IFT46 gene (). Numerous wild-type swimmers with full-length flagella were recovered. Southern blotting revealed that the exogenous IFT46 gene had integrated into the genome at different sites in several of these rescued strains (, lanes 3–9), confirming that the rescued strains were independently derived. Therefore, restoration of motility was caused by incorporation of the transgene, and not caused by disruption of some other gene. Western blotting confirmed that expression of IFT46 was restored in the transformants (, lanes 3–9). These results demonstrate that the short flagella phenotype of YH6 is caused by the absence of IFT46. Hereafter, this strain will be referred to as the ift46 mutant.
The ift46 mutant has unique defects in its axoneme
Although very short, ift46
flagella are longer than those of mutants with defects in the complex B proteins IFT88 (Pazour et al., 2000
) and IFT52 (Brazelton et al., 2001
), which do not form flagella beyond the transition region. Because flagella are formed in the ift46
mutant, we were able to compare them with flagella from wild-type and rescued cells by EM to identify flagellar defects associated with loss of IFT46. Serial sections of wild-type flagella have shown that the outer doublet microtubules are connected by rodlike “peripheral links” in the most proximal part of the flagella (); the rows of dynein arms begin at a level slightly more distal, but still within the flagellar collar (; Hoops and Witman, 1983
). The ift46
flagella have nine outer doublet microtubules and frequently extend to the limits of the flagellar collar or beyond (), but we never observed dynein arms in longitudinal or cross sections of these axonemes. In addition, the mutant flagella lack the projections into the lumens of the B-tubule (Hoops and Witman, 1983
) and frequently have defects in the central pair of microtubules (). In contrast to mutants with defects in the retrograde IFT motor (Pazour et al., 1999
; Porter et al., 1999
; Hou et al., 2004
), few, if any, IFT particles accumulate in the ift46
mutant flagella. It is important to note that the flagella from the rescued cells () have typical wild-type morphology with normal inner and outer dynein arms and central pair microtubules; this confirms that the ultrastructural defects seen in the mutant are caused by loss of IFT46.
To determine whether the dynein arm deficiency in the ift46
mutant is caused by degradation of dyneins within the cell body, by an inability to transport dyneins into the flagella, or by an inability to assemble them onto the axonemes, we analyzed whole cell lysates and flagella from ift46
and wild-type cells by Western blotting (). The cell lysates of the mutant contained normal levels of the outer arm dynein intermediate chain IC2 and the inner arm dynein intermediate chain IC138. Therefore, the dyneins are present in the mutant cells. However, both IC2 and IC138 were completely absent from the ift46
mutant flagella, indicating that the dyneins are not transported into the flagella. In contrast, DC2, which is a component of the outer dynein arm docking complex that is transported into the wild-type flagellum and assembled onto the doublets independently of the outer dynein arm (Wakabayashi et al., 2001
), is transported into the ift46
flagella. The presence of DC2 in the flagella provides additional evidence that the lack of dynein arms is not simply because of the short length of the mutant flagella or to a general failure to transport proteins into the flagellum. These data confirm that the ultrastructural findings that the ift46
mutant has a defect in transporting dynein arms into the flagella.
IFT complex B is unstable in the absence of IFT46
To investigate the role of IFT46 in IFT complex assembly, we examined the cellular levels of IFT complex B and A proteins in cell lysates of wild-type and ift46 cells (, lanes WT and ift46). When normalized with tubulin, the levels of complex B proteins IFT20, IFT57, IFT72, and IFT81 were greatly decreased in the mutant cells relative to the wild-type cells. The only complex B protein not reduced in the absence of IFT46 was IFT172, the level of which was the same as or greater than in wild-type cells, depending on the preparation, suggesting that the level of IFT172 is controlled independently from that of the other complex B proteins. In contrast to the decrease in most complex B proteins seen in the ift46 mutant, the levels of complex A proteins IFT139 and IFT140 were greatly increased in ift46 cells relative to wild-type cells.
Figure 5. IFT complex B is unstable in the ift46 mutant, but stabilized in the partially suppressed strain. Whole-cell lysates from wild-type cells, Supift461 cells, and ift46 cells were analyzed in Western blots probed with antibodies to several IFT particle proteins, (more ...)
To examine if these differences in protein levels were caused by changes in synthesis or stability, we used real-time PCR to measure transcript levels for several complex A and B proteins in wild-type and ift46 cells. Transcript levels in the mutant were increased by ~2.8- and ~2.0-fold for complex A proteins IFT140 and IFT139, and by ~1.8- and ~1.6-fold for complex B proteins IFT81 and IFT72, respectively. Therefore, the mutant responds to its defect by increasing the mRNA levels of at least these complex A and B proteins. The increase in complex A proteins in the mutant presumably reflects this increase in mRNA abundance. However, because the levels of most complex B proteins are drastically decreased in the mutant, even though complex B mRNA levels in general appear to be increased, it is likely that these proteins are specifically degraded in the absence of IFT46.
Complex A and B proteins are located in distinct compartments in the basal body region
To determine if the absence of IFT46 and the accompanying large decrease in most other complex B proteins affected the transport of IFT172 or complex A into or out of the flagellum, we used immunofluorescence microscopy to examine ift46 cells that were double labeled with antibodies to tubulin and IFT172 or IFT139 (). In both cases, the IFT particle proteins were concentrated around the basal bodies and in the short flagella. Thus, both proteins are transported into the flagella in the absence of IFT46. Surprisingly, however, the distributions of IFT172 and IFT139 differed from each other in the cell body, with that of IFT172 () appearing to have almost no overlap with that of IFT139 (), which was more anterior and often concentrated into two distinct lobes.
To clarify whether this difference in distribution was normal or caused by loss of IFT46, wild-type cells were double labeled with antibodies to IFT46 and IFT172 or IFT139. In most cases, IFT46 and IFT172 colocalized precisely with each other in the peribasal body region (), which is consistent with the other evidence that IFT46 is a complex B protein. In contrast, although IFT139 colocalized with IFT46 at the extreme apical end of the cell, the labeling of IFT46 almost always extended more posteriorly than that of IFT139 (). This is the first observation that complex A and B proteins differ in their distribution, and indicates that the complexes, or at least a subset of them, are not physically associated in the cell body. This, together with the results for the ift46 mutant, also shows that in the absence of IFT46, the colocalization of IFT172 with complex A proteins at the extreme apical end of the cell is lost.
Our observation that IFT172 was transported into the short flagella of the ift46 mutant () raised the question of whether IFT172 was being transported into the flagella independently of other complex B proteins or in association with incomplete complex B particles, possibly assembled from the small amount of complex B proteins still present in the mutant. To address this question, we used immunofluorescence microscopy to examine the distribution of another complex B protein, IFT57, the levels of which are greatly reduced in the mutant. Like IFT172, the residual IFT57 was transported into the short flagella of the ift46 mutant (). These results support the hypothesis that a small number of incomplete complex B particles assemble from the residual complex B proteins and are capable of being transported into the flagellum in the absence of IFT46. The resulting low level of IFT may account for the ability of the ift46 cells to assemble their short flagella.
Loss of IFT46 is specifically correlated with loss of the outer dynein arm in a partially suppressed strain
ift46 cells are completely nonmotile. However, on one occasion, swimming cells were observed in an unaerated, stationary phase culture of ift46 cells. Cells from this culture were cloned, and a partially suppressed strain, Supift461, was isolated. Supift461 cells are usually nonmotile when grown in M media with aeration, but are stimulated to form flagella of variable length () and swim with a slow jerky movement in the absence of aeration. The suppressed phenotype is the result of a rare spontaneous mutation that allows transcription of the 3′ end of the IFT46 gene (see next section).
Figure 7. The partial suppressor of ift46 has defects in assembly of the outer dynein arm, but not inner dynein arm I1. (A) Cells were double labeled either with antibodies to outer dynein arm heavy chain α (a, d, and g) and tubulin (b, e, and h), or with (more ...)
To elucidate the effect of the partial suppressor mutation on IFT46 and other IFT particle proteins, the levels of the proteins in stimulated Supift461, wild-type, and ift46 cells were compared by Western blotting. In the Supift461 cells, complex B proteins IFT20, IFT57, IFT72, and IFT81 were increased to a level between those of ift46 and wild-type cells (), whereas the levels of the complex A proteins IFT139 and IFT140 were decreased to a level between those of ift46 and wild type. Importantly, IFT46 is still undetected in the suppressed strain. This result indicates that the partial suppression of ift46 involves an increased stability of IFT complex B in the absence of full-length IFT46. It is possible that a C-terminal fragment of IFT46 is expressed in Supift461 cells and incorporated into complex B, thereby stabilizing it. Such a fragment would not be detected by our antibody to the N terminus of IFT46.
The slow, jerky swimming of Supift461 is typical of outer dynein arm mutants. Therefore, we used immunofluorescence microscopy to check for the presence of outer and inner dynein arms in Supift461 flagella. No outer arm dynein was detected using an antibody to the α heavy chain of outer arm dynein (). In contrast, labeling of Supift461 flagella by an antibody to inner arm dynein I1 intermediate chain IC138 was normal (). These results show that transport into the flagellum of inner arm dynein I1, but not outer arm dynein, is restored in the suppressed strain.
To determine the extent to which the ultrastructural defects of ift46 were restored in the partially suppressed strain, Supift461 cells and flagella were examined by electron microscopy (). The inner arms, radial spokes, and central microtubules were present and appeared normal. However, no outer dynein arms were observed. Therefore, the suppressor strain assembles flagella that lack the outer dynein arm but appear normal in every other way. Western blotting showed that the levels of both outer arm dynein and outer dynein arm docking complex proteins, as represented by IC2 and DC2, respectively, were normal in Supift461 cells (). Thus, the inability to transport and assemble outer arms in the flagella is not simply caused by an absence of these components from the cell cytoplasm. These results indicate that IFT46 is specifically needed to transport outer dynein arm components into the flagellum. The inner dynein arm and central pair defects observed in the ift46 mutant are likely attributable to a more general deficiency in IFT caused by the reduced number of complex B particles.
The 3′ end of the IFT46 gene is transcribed in Supift461 cells
Analysis by PCR revealed that the suppressor mutation involved a rearrangement or deletion somewhere in the region between the 3′ end of the inserted NIT1 gene and the seventh intron of the IFT46 gene (unpublished data). To determine if this mutation caused a change in the transcription of the IFT46 gene, wild-type, ift46, and Supift461 cells were examined by real-time PCR using primer pairs designed to assay for the presence of the 5′ end, middle, and 3′ end of the IFT46 mRNA (). In wild-type cells, all three regions were detected. In the ift46 mutant, only the 5′ end was detected, indicating that the 5′ end of the gene is transcribed, but a full-length mRNA is not made. Because our antibody to the N terminus of IFT46 did not detect a product, it appears that the truncated mRNA is not translated into a stable protein. This provides further evidence that ift46 is a null allele. In Supift461 cells, both the 5′ and the 3′ end, but not the middle, were reproducibly detected. Therefore, the suppressor mutation results in transcription, and possibly translation, of the 3′ end of the IFT46 gene. Our antibody would not detect a product containing the C-terminal end of IFT46, but lacking its N-terminal end. However, we can rule out the possibility that the suppressor mutation results in translation of the N-terminal part of IFT46 fused with the C-terminal part of IFT46, because our antibody did not detect any product in Supift461 cells. Transcripts encoding the 3′ end of the IFT46 gene were detected in Supift461 cells in both the presence and absence of aeration, so the suppressor mutation, not stress, causes transcription of the 3′ end of the gene.
Figure 8. The 3′ end of the IFT46 gene is expressed in the suppressor, but not in the ift46 mutant. RT-PCR was performed with no added DNA (control, C), or cDNA isolated from wild-type cells (WT), ift46 cells without aeration (ift46s), ift46 cells with (more ...)