Subcellular distribution of 14‐3‐3 proteins in the unicellular green alga Chlamydomonas reinhardtii

A polyclonal antibody was raised against a recombinant Chlamydomonas 14-3-3-beta-galactosidase (beta-Gal) fusion protein and characterized for its epitope specificity towards the corresponding Chlamydomonas 14-3-3 protein by scan-peptide analysis. This antibody recognized four Chlamydomonas polypeptides with apparent molecular masses 32, 30, 27, and 24 kDa, which also reacted with the antiserum depleted of anti-(Escherichia coli beta-Gal) IgG, but not with the corresponding preimmune serum or the antiserum preincubated with purified 14-3-3 proteins. Western-blot analyses performed with the antibody depleted of anti-(beta-Gal) IgG revealed that more or less pronounced levels of 14-3-3 proteins were present in all subcellular fractions of Chlamydomonas reinhardtii except the nuclei. The highest levels of 14-3-3 protein were observed in the cytosol and microsomal fraction. The 30-kDa isoform was predominant in the cytosol, whereas the 27-kDa isoform was prevalent in the microsomes. When microsomal membranes were separated by sucrose-density-gradient centrifugation, Western-blot analysis revealed distinct patterns of 14-3-3 isoforms in the endoplasmic reticulum, dictyosome, and plasma membrane fractions identified by marker enzyme activities. These findings indicate that the four 14-3-3 proteins of C. reinhardtii differentially interact with endoplasmic reticulum, dictyosomes, and plasma membrane.