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

• Published in: European journal of biochemistry Vol. 268; no. 24; pp. 6449 - 6457
• Main Authors: Voigt, Jürgen, Liebich, Ines, Kieß, Michael, Frank, Ronald
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Science Ltd 01-12-2001
• Subjects: 14-3-3 protein; 14-3-3 Proteins; Amino Acid Sequence; Animals; Blotting, Western; Chlamydomonas reinhardtii; Chlamydomonas reinhardtii - metabolism; cytosol; dictyosomes; Electrophoresis, Polyacrylamide Gel; isoforms; membranes; Molecular Sequence Data; Recombinant Proteins - chemistry; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Homology, Amino Acid; Subcellular Fractions - metabolism; Tyrosine 3-Monooxygenase - chemistry; Tyrosine 3-Monooxygenase - isolation & purification; Tyrosine 3-Monooxygenase - metabolism
• ISSN: 0014-2956; 1432-1033
• DOI: 10.1046/j.0014-2956.2001.02593.x

A polyclonal antibody was raised against a recombinant Chlamydomonas 14‐3‐3–β‐galactosidase (β‐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 coliβ‐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‐(β‐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.