Puromycin is incorporated into regenerating flagella of Chlamydomonas reinhardtii as an indicator of nascent flagellar proteins
The unicellular alga Chlamydomonas reinhardtii assembles flagella after pH shock-induced deflagellation, in which process the cell vigorously synthesizes flagellar proteins. Previously, newly synthesized flagellar proteins were chased using radioisotopes. Here, we have developed an alternative, non-radioactive method using the surface sensing of translation (SUnSET) assay, an assay that takes advantage of the incorporation of the antibiotic puromycin into newly synthesized peptides. Just after deflagellation, puromycin-labeled proteins increased dramatically in the cytoplasm and newly assembled flagella. Axonemal incorporation of newly synthesized proteins occurred from the distal tip of the flagellum. Cycloheximide, a protein synthesis inhibitor, almost completely prevented the cellular and flagellar incorporation of puromycin, although, as reported previously, it allowed reassembly of half-length flagella from the cytoplasmic "flagellar precursor" proteins. In contrast to wild-type cells, a cycloheximide-resistant mutant act2 produced nearly full-length flagella containing puromycin-labeled proteins even in the presence of cycloheximide. These and other results demonstrate that the SUnSET method serves as a powerful tool for visualizing flagellar incorporation of nascent proteins in Chlamydomonas.