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

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 α-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).CILIA and flagella are microtubule-based organelles that are found on most mammalian cells. They provide motility to cells and participate in many sensory processes. Defects in or loss of cilia/flagella cause a variety of human diseases that include polycystic kidney disease, retinal degeneration, infertility, obesity, respiratory defects, left–right axis determination, and polydactyly (Fliegauf et al. 2007). Mouse mutants demonstrate that cilia are essential for viability, neural tube closure, and bone development (Eggenschwiler and Anderson 2007; Fliegauf et al. 2007). Cilia and flagella are also present in protists, algae, moss, and some fungi.The assembly and maintenance of cilia and flagella require intraflagellar transport (IFT) (Kozminski et al. 1995). IFT involves the movement of 100- to 200-nm-long protein particles from the basal body located in the cell body to the tip of the flagella using the heterotrimeric kinesin-2 (anterograde movement) (Kozminski et al. 1995) and movement back to the cell body (retrograde movement) using the cytoplasmic dynein complex (Pazour et al. 1999; Porter et al. 1999). IFT particles change their direction of movement as well as their size, speed, and frequency at the ends of the flagella as they switch from anterograde to retrograde movement (Iomini et al. 2001). Biochemical isolation of IFT particles reveals that they are composed of at least 16 proteins and that these particles can be dissociated into two complexes in vitro by changing the salt concentration (Cole et al. 1998; Piperno et al. 1998). Recent genetic and bioinformatics analysis adds at least 7 more proteins to the IFT particle (Follit et al. 2009) (Eggenschwiler and Anderson 2007).

TABLE 1

Proteins and gene names for the intraflagellar transport particles in Chlamydomonas, C. elegans, and mouse

Protein	Motif	Chlamydomonas gene	C. elegans gene	Mouse gene	References to worm and mouse genes
Complex A
IFT144	WD	FLA15
IFT140	WD	—	che-11		Qin et al. (2001)
IFT139	TRP	FLA17	dyf-2	THM1	Efimenko et al. (2006); Tran et al. (2008)
IFT122	WD	—	IFTA-1		Blacque et al. (2006)
IFT121	WD	—	daf-10		Bell et al. (2006)
IFT43		—
Complex B
IFT172	WD	FLA11	osm-1	Wimple	Huangfu et al. (2003); Pedersen et al. (2005); Bell et al. (2006)
IFT88	TRP	IFT88	osm-5	Tg737/Polaris	Pazour et al. (2000); Qin et al. (2001)
IFT81	Coil	—	ift-81	CDV1	Kobayashi et al. (2007)
IFT80	WD	—	che-2	Wdr56	Fujiwara et al. (1999)
IFT74/72	Coil	—	ift-74	Cmg1	Kobayashi et al. (2007)
IFT57/55	Coil	—	che-13	Hippi	Haycraft et al. (2003)
IFT54	Microtubule binding domain MIP-T3	—	dyf-11	Traf3IP1	Kunitomo and Iino (2008); Li et al. (2008); Omori et al. (2008); Follit et al. (2009)
IFT52	ABC type	BLD1	osm-6	Ngd2	Brazelton et al. (2001); Bell et al. (2006)
IFT46		IFT46	dyf-6		Bell et al. (2006); Hou et al. (2007)
IFT27	G protein	—	Not present	Rabl4
IFT25	Hsp20	—	Not present	HSP16.1	Follit et al. (2009)
IFT22	G protein	—	IFTA-2	Rabl5	Schafer et al. (2006)
IFT20	Coil	—			Follit et al. (2006)
FAP22	Cluamp related protein	—	dyf-3	Cluamp1	Murayama et al. (2005); Follit et al. (2009)
DYF13		—	dyf-13	Ttc26	Blacque et al. (2005)

Open in a separate window—, no mutant found to date in Chlamydomonas.A collection of temperature-sensitive mutant strains that fail to assemble flagella at the restrictive temperature of 32° was isolated in Chlamydomonas (Huang et al. 1977; Adams et al. 1982; Piperno et al. 1998; Iomini et al. 2001). Analysis of the flagella at 21° permits the measurement of the velocity and frequency of IFT particles in the mutant strains. This analysis suggested that assembly has four phases: recruitment to the basal body, anterograde movement (phases I and II), retrograde movement, and return to the cytoplasm (phases III and IV) (Iomini et al. 2001). Different mutants were classified as defective in these four phases. However, because different alleles of FLA8 were classified as defective in different phases (Iomini et al. 2001; Miller et al. 2005), we combined mutants with IFT defects into just two classes. The first group (phases I and II) includes mutant strains that show decreased anterograde velocities, a decreased ratio of anterograde to retrograde particles, and an accumulation of complex A proteins at the basal body. This group includes mutations in the FLA8 and FLA10 genes, which encode the two motor subunits of kinesin-2 (Walther et al. 1994; Miller et al. 2005), as well as mutations in three unknown genes (FLA18, FLA27, and FLA28). The second group includes mutant strains that show the reciprocal phenotype (phases III and IV); these phenotypes include decreased retrograde velocities, an increased ratio of anterograde to retrograde particles, and an accumulation of complex B proteins in the flagella. With the exception of the FLA11 gene, which encodes IFT172, a component of complex B (Pedersen et al. 2005), the gene products in this class are unknown (FLA2, FLA15, FLA16, FLA17, and FLA24). One might predict that mutations in this group would map to genes that encode complex A or retrograde motor subunits. Interestingly, IFT particles isolated from fla11, fla15, fla16, and fla17-1 flagella show depletion of complex A polypeptides (Piperno et al. 1998; Iomini et al. 2001). The inclusion of IFT172 in this class is explained by the observations that IFT172 plays a role in remodeling the IFT particles at the flagellar tip to transition from anterograde to retrograde movement (Pedersen et al. 2005). The remaining mutant strains do not show obvious defects in velocities, ratios, or accumulation at 21° and may reflect a less severe phenotype at the permissive temperature or a non-IFT role for these genes.Direct interactions occur between components of complex B. IFT81 and IFT74/72 interact to form a scaffold required for IFT complex B assembly (Lucker et al. 2005). IFT57 and IFT20 also interact with each other and kinesin-2 (Baker et al. 2003). While physical interactions are being used to define IFT particle architecture, genetic interactions among loci encoding IFT components should be instructive regarding their function as well. To probe retrograde movement and its function, we have identified the gene products encoded by two retrograde defective mutant strains. They are FLA15 and FLA17 and encode IFT144 and IFT139, respectively. The genetic interactions of these loci provide interesting clues about the assembly of the IFT particles and possible physical interactions in the IFT particles.