Chapter 18 Phosphorylation of Chlamydomonas Flagellar Proteins

Abstract

Publisher Summary Phosphorylation can be studied in vivo using [ 32 P]orthophosphoric acid, but steady-state labeling of phosphoproteins in vivo depends on the coordinated actions of endogenous protein kinases and phosphatases; the extent of radioactive labeling depends on the rate at which particular phosphate groups turn over and the specific activity of the cellular ATP pool. Cilia and flagella possess a large number of phosphoproteins. In Chlamydomonas , protein phosphorylation might be involved in the regulation of axonemal motility, the photophobic response, flagellar, flagellar signaling during mating, and flagellar glycoprotein dynamic. In vivo labeling with [ 32 P] orthophosphoric acid coupled with two-dimensional polyacrylamide gel electrophoresis (PAGE) has revealed greater than 80 phosphorylated Chlamydomonas axonemal polypeptides, including components of dynein arms, radial spokes, the central pair complex, and beak projections within selected doublet microtubules. The questions that this chapter tries to find answers to are whether the in vivo and in vitro labeling with 32 P produce similar patterns of flagellar protein phosphorylation, and whether labeling varies according to different solution conditions. Significant differences were found among the patterns of flagellar membrane-matrix polypeptides phosphorylated in vitro as a function of the concentration of free Ca 2+ and major differences between the pattern of the membrane-matrix polypeptides phosphorylated in vivo and the pattern of those phosphorylated in vitro by endogenous protein kinases and phosphatases at high or low free Ca 2+ concentrations. Based on the presence of polypeptides phosphorylated in vitro that do not appear to be labeled in vivo and vice versa, it is preferable to perform in vivo phosphorylation. The experiment on in vivo labeling of Chlamydomonas flagellar proteins with [ 32 P] orthophophoric acid details the solutions, cell culture, the labeling, and flagellar fractionation. A typical experiment involving a 60-minute labeling period and 4.5xl0 9 cells yields approximately 300μl of flagellar membrane-matrix extract containing approximately 50,000 cpm/μl extract.