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. 2013 Aug 15;319(14):2275-81.
doi: 10.1016/j.yexcr.2013.06.010. Epub 2013 Jun 25.

Interaction of Mouse TTC30/DYF-1 With Multiple Intraflagellar Transport Complex B Proteins and KIF17

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Free PMC article

Interaction of Mouse TTC30/DYF-1 With Multiple Intraflagellar Transport Complex B Proteins and KIF17

Paul W Howard et al. Exp Cell Res. .
Free PMC article

Abstract

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.

Keywords: Cilia; IFT; Intraflagellar transport; Kinesin; MS; PCR; TEV; TPR; Tetratricopeptide repeat; intraflagellar transport; mass spectrometry; polymerase chain reaction; tetratricopeptide repeat; tobacco etch virus.

Figures

Figure 1
Figure 1. Expression of tagged mouse IFT57 to isolate an IFT complex from mouse pituitary cells
(A) Organization of the coding sequence of tagged mouse IFT57. The mouse IFT57 coding sequence was modified so that a calmodulin binding domain (CalB), an epitope recognized by the FLAG antibody (FLAG), 2 recognition/cleavage sites for the TEV protease (2xTEV) and 2 epitopes recognized by the AU1 antibody were added to the caroboxy-terminus of the protein. (B) Coding sequence for tagged IFT57. The first 3 amino acids and the last three amino acids of IFT57 are shown (upper case), and the sequence added to the carboxy-terminus of the protein is shown (lower case). The calmodulin binding domain, FLAG epitope, TEV recognition/cleavage sites and AU1 epitopes of the carboxy-terminal tag are indicated by underlines. (C) Expression of tagged IFT57 in the αT3-1 pituitary cell line. Cell extracts were prepared from either control αT3-1 cell or αT3-1 cells stably expressing tagged IFT57. Cell extracts were resolved by denaturing polyacrylamide gel electrophoresis, transferred to a membrane and then incubated with FLAG antiserum to detect input-tagged IFT57 or with antiserum to IFT172 to detect endogenous IFT172. To assay for formation of an IFT complex, the tagged-IFT57 was isolated by immunoprecipitation with FLAG monoclonal antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with antibody to IFT172 to detect co-immunoprecipitated IFT172. (D) Identification of proteins interacting with tagged IFT57 in pituitary cells. Whole cell extracts were prepared from control αT3-1 cells or αT3-1 cells expressing tagged IFT57. The cell extracts were incubated with AUI antibody immobilized on agarose beads. After washing of the beads, the bound proteins were eluted by digestion with TEV protease and the eluate incubated with FLAG antibodies immobilized on agarose beads. After washing, bound proteins were eluted with FLAG peptide and resolved by denaturing polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250. Stained bands that appeared to be substantially more intense in the sample from the tagged IFT57 cells were cut from the gel and subject to trypsin digestion and mass spectrometry. Several known IFT proteins were identified (labeled to the right of the gel lanes) and one 70 kDa protein. Some bands were not determined (n.d.).
Figure 2
Figure 2. The tetratricopeptide repeat-containing proteins TTC30A1, TTC30A2 and TTC30B interact with IFT52
(A) HEK293 cells were transfected with expression vectors for AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B and FLAG tagged-IFT52 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of the tagged-TTC30 isoforms. A non-specific band was detected in all lanes. (B) Input expression of FLAG-IFT52 was detected by immunoblotting with FLAG monoclonal antibody. (C) To detect interaction of AU-tagged TTC30 isoforms with IFT52, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30A1, AU1-TTC30A2 or AU1-TTC30B. (D) Imunoblotting of cell extracts with antibody to ERK-1 as a loading control.
Figure 3
Figure 3. Interaction of TTC30B with multiple IFT proteins in a cell-free, in vitro system
Coding sequences for tagged proteins were prepared by PCR and fused to a promoter for bacteriophage SP6 polymerase. The promoter-tagged protein DNA was used to direct protein synthesis in a cell-free coupled transcription-translation system from wheat germ. The in vitro reactions contained either coding sequences for AU1-tagged TTC30B or FLAG-tagged IFT protein as indicated. (A) To analyze protein expression, aliquots of the in vitro reactions were resolved by denaturing gel electrophoresis and immunoblotting with antibody to FLAG to detect the input expression of IFT proteins or with AU1 antibody to detect input expression of TTC30B. non-specific band was detected in all lanes and serves as a loading control. (B) To detect interaction of TTC30B with IFT proteins, FLAG-tagged IFT proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitate, AU1-tagged-TTC30B.
Figure 4
Figure 4. Interaction of TTC30B and KIF17 with IFT52 and IFT57 in cells
(A) HEK293 cells were transfected with expression vectors for AU1-tagged TTC30B or HA-tagged KIF17 and FLAG tagged-IFT52 or FLAG-tagged IFT57 as indicated. At 20 hours after transfection, whole cell extracts were prepared. (A) Samples were analyzed by denaturing gel electrophoresis and immunoblotting with antibody to AU1 to detect the input expression of TTC30B. (B) To detect interaction of TTC30B with IFT52 or IFT57, FLAG-tagged proteins were isolated by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with AU1 antibody to detect co-immunoprecipitated AU1-TTC30B. (C) Input expression of HA-KIF17 was detected by gel electrophoresis and immunoblotting with HA antibody. (D) Interaction of KIF17 with the IFT complex was analyzed by immunoprecipitation with FLAG antibody and the immunoprecipitate was analyzed by gel electrophoresis and immunoblotting with HA antibody to detect co-immunoprecipitated HA-KIF17.

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