Phosphorylation of nuclear and flagellar basal apparatus proteins during flagellar regeneration in Chlamydomonas reinhardtii.
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.
Harper
J D
JD
Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905.
Sanders
M A
MA
Salisbury
J L
JL
D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005407:Flagella / Q000502:physiology*; D005455:Fluorescent Antibody Technique; D036341:Intercellular Signaling Peptides and Proteins; D008970:Molecular Weight; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010455:Peptides; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D015800:Protozoan Proteins / Q000378:metabolism; D012038:Regeneration; D014862:Wasp Venoms / Q000494:pharmacology
D036341:Intercellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D010455:Peptides; D010750:Phosphoproteins; D015800:Protozoan Proteins; D014862:Wasp Venoms; C037500:mastoparan