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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cilia / metabolism
  • HEK293 Cells
  • Humans
  • Kinesin / metabolism*
  • Mice
  • Molecular Sequence Data
  • Multiprotein Complexes / metabolism
  • Pituitary Gland / cytology
  • Protein Binding
  • Proteins / genetics
  • Proteins / metabolism*
  • Receptors, Cell Surface / chemistry
  • Receptors, Cell Surface / metabolism*

Substances

  • KIF17 protein, mouse
  • Multiprotein Complexes
  • Proteins
  • Receptors, Cell Surface
  • TTC30 protein, mouse
  • Kinesin