Characterization of Fmg-1B, the major Chlamydomonas flagellar membrane glycoprotein

Robert A. Bloodgood and Anthony J. Spano

Dept. of Cell Biol. and Dept. of Biol., Univ. of Virginia, Charlottesville, Virginia, 22908, USA

The major C. reinhardtii flagellar membrane protein migrates as a high molecular weight doublet. Mouse monoclonal antibodies recognizing both these bands were used to screen a cDNA expression library, yielding a partial cDNA used to identify genomic BACs from which two genes (FMG-1A and FMG-1B) were completely sequenced. FMG-1A and FMG-1B encode predicted protein sequences with 57% identity (90% identity in the C-terminal 1000 amino acids). Based on peptide sequences from the Chlamydomonas proteome and EST sequences, only FMG-1B is expressed in vegetative cells; deflagellation induces a 5 fold increase in expression. The FMG-1B transcript contains a single open reading frame encoding a protein of 4150 amino acids (410 kD) plus the signal sequence. Observations supporting this as the native protein found in the two gel bands include: 1) sequences obtained from the native protein are perfect matches for 50% of the predicted sequence, 2) antibodies made to predicted peptide sequences recognize the native protein and 3) the predicted amino acid composition is an excellent match to the amino acid composition of the bands excised from SDS gels. The Fmg-1B protein has a 27 amino acid N-terminal signal sequence, a single transmembrane domain and a 17 amino acid C-terminal cytoplasmic domain. Motifs in the ectodomain of the protein include: 1) 31 potential N-glycosylation sites, 2) a 20 amino acid proline-rich domain containing PSPP repeats and 3) an ATP/GTP-binding P-loop domain. Biochemical and cell biological data suggest that Fmg-1B is required for sensory and motor events associated with substrate adhesion and whole cell gliding motility. Supported by NSF MCB-9904916.

e-mail address of presenting author: rab4m@virginia.edu