Sec7 GEFs of Tetrahymena thermophila: Domain analysis and cellular localization

Abstract

Sec7 GEFs are known to activate small G proteins of the ARF (ADP ribosylation factors) family, which act to regulate cellular functions such as vesicular trafficking, microtubule dynamics and development. In 1999, a guanine nucleotide exchange factor (GEF) was identified in Paramecium from a screen for ciliary proteins and was named PSec7 (Nair et al., 1999). The Paramecium PSec7 antibody was found to recognize a putative GEF protein in Tetrahymena thermophila, Gef1p (GEF1), which localizes to the cilia and macronucleus in immunofluorescence, immunoEM and western blot experiments. PCR methods and database analysis were used to identify a Tetrahymena protein 2053 AA in length, presumably GEF1. Two additional GEFs of the GBF/BIG Sec7 subfamily (GEF2, GEF3) as well as two other Sec7 containing GEFs that belong to a subfamily unique to alveolates (the TBS subfamily) have been identified in the Tetrahymena genome database. In addition to the canonical motifs representative of GBF/BIG subfamily members (DCB, HUS, SEC7 and HDS domains), GEF1 possesses both ciliary and nuclear targeting sequences, truncated IQ-like motifs as well as putative PH domains, homologous to similar domains in Psec7. Ciliary targeting sequences are not found in GEF2 and GEF3. Upregulation of mRNA for GEF1 was observed via RT-PCR following deciliation and subsequent regrowth of cilia in Tetrahymena. Constructs were made to incorporate a hemagglutinin (HA) sequence on to these proteins in order to determine the localization of GEF1 and GEF2. Cells transformed and selected to produce a truncated GEF1-HA showed a weak ciliary phenotype. The truncated protein localized appropriately to both somatic and oral cilia and not to the macronucleus. GEF2-HA was determined to localize to the basal bodies with no ciliary and limited macronuclear localization, which suggests that GEF1 is probably the protein being recognized by the PSec7 antibody. The function of GEF1 in IFT transport of ciliary membrane proteins is discussed.