Cloning and Sequencing of Chlamydomonas IFT88
To learn more about the structure and function of the proteins that make up the IFT particle, we cloned and sequenced the IFT88 protein, formerly known as p88 (Cole et al. 1998
). To do this, Chlamydomonas
IFT particles were purified from the matrix of isolated flagella by sucrose density gradient centrifugation and two-dimensional gel electrophoresis. IFT88 was cleaved by trypsin and two internal peptides were microsequenced (Cole et al. 1998
), yielding the sequences AATNLAFLYFLEGETDQADKYSEMALK and SLFNEAAGIDPYCVEAIYNLGLVSQR. Degenerate PCR primers were designed from these sequences and used to amplify a fragment of genomic DNA. A cDNA library was screened with the genomic fragment and the resulting clones were sequenced by primer walking. Southern blots indicated that there is only one copy of the IFT
88 gene in the Chlamydomonas
Sequence analysis showed that the IFT
88 cDNA contains a 2,346-nucleotide open reading frame that is predicted to encode an 86.3-kD protein ( a) with a pI of 5.87. Perfect matches to both IFT88 tryptic peptides are found in the open reading frame of this cDNA, rigorously confirming that these clones encode the Chlamydomonas
IFT88 protein. No discernible motifs were identified within the sequence except for the presence of 10 tetratricopeptide repeats (TPR) ( b). TPRs are degenerate 34–amino acid repeats (Lamb et al. 1995
), present in tandem arrays of 3–16 U that are predicted to form amphipathic helices (Hirano et al. 1990
). The first three TPR motifs are found closely spaced between amino acid residues 185–294. The other seven TPR motifs occur without spacing between amino acid residues 441–676 ( c).
Sequence and structure of the Chlamydomonas
IFT88 protein. (a) Chlamydomonas
IFT88 is homologous to the mouse and human Tg737 proteins. The sequences of the Chlamydomonas
(Cr_IFT88, accession number AF298884), mouse (Mm_Tg737, accession number AAB59705), and human (Hs_Tg737, accession number AAA86720) proteins were aligned using ClustalW (Thompson et al. 1994
). (*) Complete conservation; :, highly conserved substitutions; and (.) semiconservative substitutions (below the alignment). (b) IFT88 contains 10 TPR repeats. Residues matching the TPR consensus sequence (bottom) are indicated by bold font. (c) The 10 TPR repeats (shaded boxes) are organized in a group of three in the NH2
-terminal half of the protein and a group of seven in the COOH-terminal half of the protein.
Chlamydomonas IFT88 Is Homologous to a Mouse Polycystic Kidney Disease Gene
Blast searches with the Chlamydomonas IFT88 protein sequence indicate that it is very similar to the mouse (41% identical; BLAST E=e-148) and human Tg737 (40% identical; BLAST E=e-146) proteins. Mice with defects in Tg737 have severe polycystic kidney disease and die within a few weeks of birth. The protein also is homologous to proteins predicted by ESTs from zebra fish and swine and fragments of preliminary C. elegans and Drosophila melanogaster genomic sequence. IFT88 and Tg737 are likely to be functionally equivalent orthologs as the similarity between the Chlamydomonas and mammalian proteins is robust and distributed over the entire coding region and not just within the TPR domains ( a). 40% identity is very typical of the amount of similarity seen between other Chlamydomonas and mammalian orthologs that encode flagellar proteins (Pazour, G.J., B.L. Dickert, and G.B. Witman, manuscript in preparation).
IFT88 Is Required for Flagellar Assembly
To learn more about the function of IFT88 in cells, we searched our collection of Chlamydomonas
insertional mutants (Pazour et al. 1995
, Pazour et al. 1998
; Pazour and Witman 2000
) for a cell line with a defect in this gene. The insertional mutants were made by transforming cells with DNA carrying a selectable marker. In Chlamydomonas
, transforming DNA is integrated randomly throughout the genome and disrupts genes at the site of integration. DNA was isolated from ~400 insertional mutants having behavioral or motility defects and was screened by Southern blotting using a fragment of IFT
88 genomic DNA as a probe. One cell line (V79) was identified that had an insertion in the IFT
88 gene ( a). The fact that the single hybridizing band in wild-type cells was split into two bands in the mutant indicated that the selectable marker integrated into the gene within the region covered by the probe and did not result in a large deletion of the genome at the site of integration. The mutant allele was termed ift
Figure 2 Phenotype of the ift88-1 mutant cells. (a) Southern blot showing that the V79 cell line has an insertion in the IFT88 gene. DNA was isolated from wild-type and V79 cells, digested with PstI, and analyzed by Southern blotting with a 365-bp fragment of (more ...)
88-1 cells grew at the same rate as wild-type cells, indicating that IFT88 is not required for processes essential for growth or cell division ( b). However, in contrast to wild-type cells that normally have two ~10-μm long flagella extending from the anterior end of the cell body, the ift
88-1 cells completely lack flagella ( c). Electron microscopic analysis of these cells showed that the basal bodies were structurally normal but the flagella did not extend beyond the transition zone (, arrows). In some cells, the membrane covering the flagellar tips was tightly apposed to the microtubules with no material between them and the membrane. In other cells, the flagellar stubs were slightly swollen and contained fragments of microtubules in random orientations. However, in contrast to the IFT mutants fla
14 (Pazour et al. 1998
) and dhc
1b (Pazour et al. 1999
), no accumulation of IFT particles was observed in any of the flagellar stubs.
Figure 3 Ultrastructure of the ift88-1 flagella. The flagella on ift88-1 mutant cells are very short and the microtubules do not extend beyond the transition zone (arrows). The microtubules in wild-type cells start at the basal body, extend through the transition (more ...)
To determine the effect of the lack of IFT88 on the IFT particle and the IFT motors, we examined whole-cell extracts by Western blotting (). The IFT particle is made up of two large complexes. Complex A is composed of four to five proteins and includes IFT139, shown in . Complex B is composed of IFT88 and 10 other proteins including IFT172, IFT81, and IFT57, shown in . The complex A protein IFT139 is enriched in the mutant, suggesting that the gene may be upregulated in the mutant cells. The mutation has little or no effect on the levels of complex B proteins IFT172 and IFT81, but causes a significant decrease in IFT57, another complex B protein. The cellular levels of the IFT motors FLA10 kinesin-II and DHC1b are not affected by the ift88 mutation.
Figure 4 Western blot showing the effect of the ift88-1 mutation on the levels of IFT motor and particle proteins. Equal amounts of whole-cell extracts of ift88-1 (3276.2) and wild-type cells (CC124) were separated by SDS-PAGE, transferred to membrane and probed (more ...)
To be certain that the flagellar assembly defect is caused by the mutation in IFT88 and is not the result of another mutation elsewhere in the genome, we transformed the mutant cells with BAC clones carrying the IFT88 gene. Three independent BAC clones (40-B3, 24-F2, and 27-M3) complemented the flagellar defect. The complemented cell lines swam like wild-type cells and had IFT. One of the rescued cell lines was crossed to a wild-type cell line and 26 tetrads were dissected. All four products of one tetrad and a single product of the remaining 25 tetrads were analyzed by Southern blotting. Because the transformed copy of the IFT88 gene inserted at a site unlinked to the original locus, the inserted DNA segregated independently from the original gene, allowing offspring to carry zero, one, or two copies of the wild-type gene. Cells that carry at least one copy of wild-type IFT88 have normal flagella and motility, whereas those that carry no copies of wild-type IFT88 lack flagella and are nonmotile (). These data indicate that the flagellar defect is tightly linked to the ift88 mutation and is almost certainly the result of it.
Figure 5 Presence of the IFT88 gene correlates with the wild-type phenotype in meiotic products. Strain V79 was transformed with a BAC clone containing the IFT88 gene. Transformed cells recovered the ability to swim and were enriched by taking inoculi from the (more ...)
Primary Cilia in the Kidney of Tg737 Mice Are Shorter than Normal
Primary cilia are present on many cells in the mammalian body (Wheatley 1995
; Wheatley et al. 1996
), and are particularly well developed in the kidney (Andrews and Porter 1974
). Inasmuch as Chlamydomonas
IFT88 is necessary for assembly of flagella and is homologous to mammalian Tg737, and because a defect in mouse Tg737 leads to kidney disease, it was of great interest to determine whether the defect in Tg737 affected formation of the primary cilia in the kidney. In wild-type rats, the cilia are ~2.5-μm long and are found in the proximal tubule, the loop of Henle, the distal tubules, and the collecting ducts (Andrews and Porter 1974
). In wild-type mice, these cilia are <5-μm long and similarly distributed (Flood and Totland 1977
We obtained the hypomorphic Tg737-mutant mice from Oak Ridge National Laboratory and examined the kidneys of 4- and 7-d–old pups by scanning electron microscopy. Numerous monociliated cells were observed in the kidneys of both wild-type ( a, +/+) and homozygous mutant ( a, −/−) mice, but the cilia in the mutant kidneys were much shorter. To quantify this difference, the cilia lengths were measured from scanning electron micrographs taken from the tubules distal to the proximal tubule ( and ). The proximal tubule was avoided because it contains a thick brush border that can obscure a micron or more of cilia length. The tubules distal to the proximal tubule have only sparse microvilli that do not obscure cilia, and the cilia in these regions are uniform in length (Andrews and Porter 1974
). These cilia in wild-type mice were 3.1 ± 1.4 and 3.5 ± 1.7 μm at 4 and 7 d, respectively, whereas these cilia in the mutant mice were 1.0 ± 0.6 and 1.3 ± 0.6 μm at 4 and 7 d, respectively. These values represent minimum lengths as it is difficult to accurately measure cilia that are lying at different angles in the tubules. However, the differences are quite large and are significant at the >99% level. Thus, Tg737 plays an essential role in assembly of the primary cilium in the mouse, just as IFT88 is essential for flagellar assembly in Chlamydomonas.
Primary cilia in the kidney of Tg737 mutant mice are shorter than normal. (a) Kidneys from 4-d-old pups were fixed with glutaraldehyde, freeze fractured, metal impregnated, and examined by scanning electron microscopy. Numerous cilia were found on the epithelial cells in the tubules and collecting ducts of the wild-type mice (+/+). Cilia were also found in the homozygous mutant (−/−) pups, but they were usually <2-μm long and most were only short stubs. (b) Cilia length in kidneys of 4-d-old mice. Cilia were measured in scanning electron micrographs of tubules located distal to the proximal tubule. Wild-type cilia averaged 3.1 ± 1.4 μm (n = 50), whereas the mutant cilia averaged 1.0 ± 0.6 μm (n = 50). (c) Cilia length in kidneys of 7-d-old mice. The cilia lengths were measured in scanning electron micrographs of tubules located distal to the proximal tubule. Wild-type cilia averaged 3.5 ± 1.7 μm (n = 50), whereas the mutant cilia averaged 1.3 ± 0.6 μm (n = 50).