The Journal of Cell Biology, Vol 122, 877-886, Copyright © 1993 by The Rockefeller University Press

ARTICLES

Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii

JD Harper, MA Sanders and JL Salisbury
Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905.

The antiphosphoprotein monoclonal antibody MPM-2 was used to investigate protein phosphorylation during flagellar regeneration in Chlamydomonas reinhardtii. MPM-2 recognizes a phosphorylated epitope and detects several Chlamydomonas proteins by Western immunoblot analysis. Two MPM-2 reactive proteins (34 and 90 kD) increase in Western immunoblot intensity after flagellar excision and decrease in intensity during flagellar regeneration. Immunofluorescence and immunogold labeling revealed MPM-2 staining within the nucleus, especially towards the nuclear periphery, the flagellar basal apparatus, and the nucleus-basal body connector after flagellar excision. Comparison of MPM-2 reactivity in wild-type cells and in the mutant bald-2, which lacks functional basal bodies, demonstrates that the 34-kD protein is localized in the nucleus and the 90-kD protein is localized in the flagellar basal region. MPM-2 reactivity is observed in cells competent for flagellar regeneration. However, when cells were treated with the kinase inhibitor, staurosporine, MPM-2 reactivity did not increase after flagellar excision and flagellar regeneration was impaired. These observations suggest that phosphorylation of the 34- and 90-kD proteins may be important for flagellar regrowth. Possible roles for phosphorylation in flagellar regeneration include transcriptional activation and transport of flagellar precursors to the base of the growing flagella.
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