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. 2009 Nov;183(3):885-96.
doi: 10.1534/genetics.109.101915. Epub 2009 Aug 31.

Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas Reinhardtii

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Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas Reinhardtii

Carlo Iomini et al. Genetics. .
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The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not alpha-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172).


F<sc>igure</sc> 1.—
Figure 1.—
Schematic diagram of segregation of heterozygosity in rescued transformants (SHIRT). SHIRT uses mapping and PCR-based markers to determine which parts of a BAC are responsible (cosegregate) for rescue of a mutant phenotype following transformation. (A) Diagram of three genes present on a BAC that come from the mutant parent (black), the polymorphic mapping strain CC-1952 (red), and the BAC (blue). Primers for PCR are generally made to the 3′-UTR of genes to be tested (arrows). (B) Bands of digested PCR products (dCAP markers) for the three genes from the three sources of DNA. The digested PCR products from the black and blue alleles cannot be distinguished from each other. (C) Three possible outcomes among nonparental ditype (NPD) tetrads in which the mutant phenotype is observed in two of the four progeny (fla and +). The pattern with gene 1 suggests that the BAC DNA is not responsible for rescue. The hybrid (black and blue) band indicates that the band is amplified from both the mutant and the BAC allele. The pattern with gene 2 is consistent with the BAC DNA providing rescue. Further proof requires additional NPD tetrads with this pattern. The pattern with gene 3 suggests that it is not integrated into the genome and therefore not responsible for rescue.
F<sc>igure</sc> 2.—
Figure 2.—
Identification of IFT144 as the gene product of the FLA15 locus. (A) Diagram of the structure of the gene. IFT144 is a WD repeat protein (Cole 2003). It also has a clathrin domain, which has been implicated in vesicle transport in the testis (Oyhenart et al. 2003), as well as an uncharacterized domain (PD303210). (B) The region of IFT144 from mouse (Mm), human (Hs), Chlamydomonas (Cr), Drosophila (Dm), C. elegans (Ce), and Giardia (Gl) that contains the cysteine that is mutated in fla15-1. This cysteine is invariant (shaded). (C) SHIRT analysis of the transgene. PCR products from a nonparental ditype are shown. The parents (fla15 + rescue; CC-1952) are in lanes 1 and 2. The four progeny are in lanes 3–6. Lanes 3 and 6 are from cells aflagellate at 32° and the band is consistent with the mutant alleles. Lanes 4 and 5 are from flagellated cells and the bands are consistent with the wild-type FLA15 allele from the CC-1952 parent and the BAC contributed transgene.
F<sc>igure</sc> 3.—
Figure 3.—
Identification of IFT139 as the gene product of the FLA17 locus. (A) Diagram of the structure of the gene. IFT139 contain multiple TRP domains that are thought to mediate short-term protein interactions and two half A TPR (HAT) domains. Shaded exons represent the three exons deleted in the fla17 alleles. (B) PCR amplification of IFT139 with primers in exons 16 and 20 (5′-ATC CGC GAG ACG CCT CTG TAC and CTG TGC GCC GCC GCG GGC GT). The wild-type product is ∼700 bp while the mutant product of fla17-1 and fla17-3 (also known as fla16-1) is ∼325 bp. (C) Alignment of protein around and including the deletion in human, mouse, Chlamydomonas, and C. elegans. The deleted region is shown in boldface type. (D) Exon 10 in the IF139 gene shows alternative splicing. In black are the shared introns before and after exon 10. Exonic sequence that is present in both splicing variants is shown in red. In green is the exon that is present in only one splice variant. The four splice donor and acceptor sites are underlined and in uppercase. The 58 amino acids encoded by the longer splice variant are shown in blue and the 28 amino acids encoded by the short splice variant are shown in purple.
F<sc>igure</sc> 4.—
Figure 4.—
Immunoblots for IFT components with flagellar extracts of wild-type and mutant strains grown at 21°. (A) Coomassie blue-stained gels of wild type, fla15, fla17, and rescued strains fla15-1∷IFT144 and fla17-1∷IFT139. (B) Immunoblots of the region around 145–130 kDa reacted with antibodies to complex A, IFT74/72, and α-tubulin. The complex A antiserum has been shown previously to recognize the IFT139 polypeptide (Iomini et al. 2001) and IFT74/72 is described in Iomini et al. (2004).
F<sc>igure</sc> 5.—
Figure 5.—
Retrograde mutant bulges contain some but not all flagellar proteins. (A and B) Phase contrast of bulges (arrowheads) on fla15-1 cells (A) and their absence in the rescued fla15-1∷IFT144 strain (B). In the mutant strain, >80% of the cells have bulges (n = 200) and no bulges were observed in the rescued strains. (C–E) Phase images of cells with bulges. (F–H) Immunofluorescence of cells with bulges. (F) p28 antibody. (G) IFT74/72 antibody. (H) α-Tubulin antibody. IFT74/72 is present in the bulges, but α-tubulin and p28 are not.
F<sc>igure</sc> 6.—
Figure 6.—
Diagram of possible interactions in the IFT particle. (A) IFT complexes A and B with kinesin-2 (in green) and cytoplasmic dynein (in red). (B) Synthetic phenotypes suggest interactions between all of the mutants with retrograde defects. (C) Noncomplementation phenotypes show more limited interactions. The fla2 mutant no longer interacts. (D) Two-copy suppression shows interactions of IFT139 and IFT144 while IFT172 shows a weak interaction with IFT144.

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