| Molecular
Cloning of an EF-hand Protein in Chlamydomonas Flagellar Radial
Spokes
Jennifer
Dienes
Mentor:
Dr. Pinfen Yang
Marquette
University
Milwaukee,
WI
Radial
spokes play a crucial role in ciliary and flagellar motility. Although
several of the radial spoke proteins are cloned, RSP7 remained to be characterized.
The 2-D signature map of Chlamydomonas radial spoke proteins (RSP) (Piperno
et al., 1981) enabled a 58 kD spot, corresponding to RSP7, to be purified
from a 2-D gel of isolated 20S radial spokes. The purified protein was
then subjected to mass spectrometry. From MS/PDS analysis as well as Database
searching, two tryptic peptides were discovered to correspond to an EST
clone containing one open reading frame and an incomplete genomic scaffold
respectively. Importantly, a ~20bp region of the EST overlapped the partial
genomic sequence after a large unknown region. This suggested that the
partial sequences found were, indeed, portions of the RSP7 cDNA and gene.
In
order to complete the protein and nucleotide sequences, RT-PCR and PCR
were both carried out, with the resulting products being cloned and sequenced.
The partial cDNA of RSP7 encodes a 48kD polypeptide. Further analysis of
this sequence using BLAST, SMART, and SupFam programs has revealed 4 to
6 calcium binding EF-hand domains, which are homologous to several other
EF-hand proteins. This sequence homology suggests that RSP7 acts as a calcium
sensor within the radial spoke. From this information, we propose that
the radial spoke mediates the calcium-induced waveform changes observed
in Chlamydomonas via the novel EF-hand protein, RSP7, in addition to the
radial spoke calmodulin (Yang et al., 2001). |
