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.
Voigt
J
J
Physiologisch-chemisches Institut, Universität Tübingen, Germany. juergen.voigt@uni-tuebingen.de
Liebich
I
I
Kiess
M
M
Frank
R
R
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism*; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D048948:14-3-3 Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase