Flagellar morphogenesis: Protein targeting and assembly in the paraflagellar rod of trypanosomes

The paraflagellar rod (PFR) of the African trypanosome Trypanosoma brucei r epresents an excellent model to study flagellum assembly. The PFR is an int raflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epit ope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in try panosomes growing a new flagellum provided an excellent marker of newly syn thesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR Moreover, we have obs erved turnover of epitope-tagged PFRA in old flagella that takes place thro ughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by imp ort or binding. This sequence was not sufficient to confer full flagellum l ocalization to a green fluorescent protein reporter. A second sequence, nec essary for the addition of PFRA protein to the distal tip, was also identif ied. In the absence of this sequence, the mutant PFRA proteins were localiz ed both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved i n all PFRA and PFRC proteins and shows homology to a sequence in the flagel lar dynein heavy chain of Chlamydomonas reinhardtii.