Intraflagellar transport (IFT) protein IFT25 is a phosphoprotein component of IFT complex B and physically interacts with IFT27 in Chlamydomonas

Abstract

Background: Intraflagellar transport (IFT) is the bidirectional movement of IFT particles between the cell body and the distal tip of a flagellum. Organized into complexes A and B, IFT particles are composed of at least 18 proteins. The function of IFT proteins in flagellar assembly has been extensively investigated. However, much less is known about the molecular mechanism of how IFT is regulated.

Methodology/principal findings: We herein report the identification of a novel IFT particle protein, IFT25, in Chlamydomonas. Dephosphorylation assay revealed that IFT25 is a phosphoprotein. Biochemical analysis of temperature sensitive IFT mutants indicated that IFT25 is an IFT complex B subunit. In vitro binding assay confirmed that IFT25 binds to IFT27, a Rab-like small GTPase component of the IFT complex B. Immunofluorescence staining showed that IFT25 has a punctuate flagellar distribution as expected for an IFT protein, but displays a unique distribution pattern at the flagellar base. IFT25 co-localizes with IFT27 at the distal-most portion of basal bodies, probably the transition zones, and concentrates in the basal body region by partially overlapping with other IFT complex B subunits, such as IFT46. Sucrose density gradient centrifugation analysis demonstrated that, in flagella, the majority of IFT27 and IFT25 including both phosphorylated and non-phosphorylated forms are cosedimented with other complex B subunits in the 16S fractions. In contrast, in cell body, only a fraction of IFT25 and IFT27 is integrated into the preassembled complex B, and IFT25 detected in complex B is preferentially phosphorylated.

Conclusion/significance: IFT25 is a phosphoprotein component of IFT particle complex B. IFT25 directly interacts with IFT27, and these two proteins likely form a subcomplex in vivo. We postulate that the association and disassociation between the subcomplex of IFT25 and IFT27 and complex B might be involved in the regulation of IFT.

Figure 1. IFT25 is a phosphoprotein component of the IFT particle.

(A). Identification and purification of IFT25 from isolated flagella extracts of Chlamydomonas. The soluble flagellar proteins isolated from flagella of the wt cells were fractionated on a 12-ml 10–25% sucrose gradient. The Coomassie blue-stained 10% SDS-PAGE gel of gradient fractions 1 (25% sucrose) to 24 (10% sucrose) is shown. IFT particle proteins peaked at the 16S fractions. The proteins highlighted in grey-lined rectangles are IFT particle complex A proteins with high molecular weights. IFT27, IFT25, and IFT22 polypeptides are highlighted with arrows. (B). Antibody α-IFT25 recognizes two bands on an immunoblot of wt whole cell extracts. (C). IFT25 is a phosphoprotein. Two bands were detected from the flagella extracts using an antibody against IFT25 protein on Western blots. After the treatment with calf intestine alkaline phosphatase (CIAP), the upper band disappeared and the intensity of the lower band was correspondingly increased. Addition of exogenous ATP elevated the level of the upper band, and the lower band showed a corresponding decreased intensity. The axonemal protein IC69 was used as a loading control.

Figure 2. IFT25 behaves similarly to complex B subunits but not complex A subunits in anterograde IFT FLA10-kinesin-II ts mutants.

Wild-type, fla10-1, fla8, and fla3-1b mutants grown in M1 medium at 18°C were incubated at 32°C for 0, 1.0 or 1.5 hours prior to deflagellation. The flagella were purified and analyzed with 10% SDS-PAGE and immunoblotting. The antibodies used in immunoblotting included those against IFT25, complex A subunits IFT139 and IFT122, complex B subunits IFT81 and IFT27, FLA10, KAP, and D1bLIC as indicated on the left. IC69 was used as a loading control.

Figure 3. IFT25, similarly to IFT complex B subunits, accumulates in three retrograde IFT ts mutants.

(A). Coomassie blue staining of the purified flagella proteins on a SDS-PAGE gel. Wild-type, fla15, fla16, and fla17-1 mutants grown in M1 medium at 18°C were incubated at 32°C for 0, 1.0 or 1.5 hours prior to deflagellation. The flagellar proteins were purified, quantified, and separated on a 10% SDS-PAGE gel with a loading of the same amount of proteins per lane. The molecular markers are labeled on the left. The incubation time is marked on the top. (B). Immunoblots derived from the SDS-PAGE gel shown in A. Flagellar proteins on the gel were transferred to a nitrocellulose membrane. The membrane was then probed with antibodies against IFT25, the complex A subunits IFT122 and IFT139, the complex B subunits IFT172, IFT81, and IFT27, FLA10, or D1bLIC, and a flagellar axonemal protein IC69 as indicated on the left. Two bands corresponding to IFT139 were detected from the flagella of fla16 and fla17-1 mutants when incubated at 32°C. (C). Two different isoforms of IFT139 were detected. The immunoblot probed with the IFT139 antibody showed that the flagella isolated from fla16 mutant contained a single IFT139 band migrating faster than that isolated from wt cells when incubated at 18°C. When the temperature was shifted to 32°C, a slower-migrating band with a size similar to IFT139 appeared.

Figure 4. IFT25 has a unique distribution pattern.

(A). Flagella and cell body distribution patterns of IFT25, IFT27, and IFT46. Fluorescent microscopy assay showed that IFT25, IFT27, and IFT46 (green) had spotted distribution along the entire length of flagella, a typical pattern of IFT proteins (A. top row). α-tubulin (red) was used to serve as a marker for basal bodies and flagella. The bottom row shows the cell body distributions of those three proteins. The scale bar is 5 µm.The details of staining at the basal bodies are enlarged and shown in B. (B). Cell body distribution patterns of IFT25, IFT27, and IFT46. Using IFT27 and IFT46 as controls, fluorescent microscopy assay showed that IFT25 (green) was localized into two regions: two bright dots comprising a region directly above each basal body, probably the transition zones (above the α-tubulin shown as red in B. top row) and a band possibly below each basal body (partially overlapped with the α-tubulin shown as red). IFT27 (green) was localized into regions that were probably the transition zones, but was not present in the proximal areas of the basal bodies (B. middle row). IFT46 (green) was found to overlap with α-tubulin (red) at the basal bodies (B. bottom row). The scale bar equals 1 µm.

Figure 5. IFT25 interacts physically with IFT27.

Purified GST-tagged IFT27 and MBP-tagged IFT25 were used for in vitro binding assay. The left panel shows that immobilized GST-IFT27 protein on the beads could retain MBP-IFT25 protein but not the control protein, MBP. The right panel shows that immobilized MBP-IFT25 protein on beads could retain GST-IFT27 protein but not the control protein, GST. The molecular marker is labeled on the left of each figure. From top to bottom for both panels, the first figure is the Coomassie blue-stained gel. The second and third figures represent the immunoblots probed with antibodies against IFT25 and IFT27, respectively. The fourth one is the immunoblots probed with antibodies against either MBP (left panel) or GST (right panel). The loading materials for each lane of the gels are shown in the tables at the top of each panel. S stands for supernatant and P for bead pellet.

Figure 6. The majority of IFT25 and IFT27 are not integrated into the IFT complex B in the cell body.

(A). Wild-type flagella extracts or cell body lysates of the bld2 mutant were fractionated on a 10–25% sucrose gradient. The fractions were then separated by SDS-PAGE. Immunoblots probed with antibodies against several IFT particle proteins as indicated to the left of the blots. Flagellar IFT25 and IFT27 were found in two peaks, a major peak at 16 S fractions, and a minor peak at much lighter fractions. IFT46, a complex B subunit, only peaked at 16S fractions. Only a portion of whole-cell IFT25 and IFT27 was integrated into the preassembled IFT complex B in the cell body. IFT particle proteins, including complex A subunit IFT139 and complex B subunits IFT172, IFT81, and IFT74.were found entirely in 16S fractions. In contrast, IFT25 and IFT27 had two peaks on the gradient. The Western blots shown for IFT25 were exposed for 1 minutes or 5 minutes to ensure that phosphorylated IFT25 was detected in the 16S fractions. (B). Ponceau S staining of the membrane showing the protein profiles of whole cells, cell bodies, and flagella. Flagellar proteins were isolated from 5×10⁶ (5×), 1.5×10⁷ (15×), and 5×10⁷ (50×) cells. Whole cell or cell body proteins were isolated from 1×10⁶ (1×), 3×10⁵ (0.3×), and 1×10⁵ (0.1×) cells. (C). The distributions of IFT25 and IFT27 proteins among whole cells, cell bodies, and flagella. The same or a duplicated membrane shown in B was probed with antibodies as indicated to the left. (D). The intensity of each lane or band stained by Ponceau S or antibodies. Photoshop CS3 software was used for the measurement.