Purification of the Chlamydomonas reinhardtii CIA5 protein, the master regulator of the Chlamydomonas Carbon Concentrating Mechanism

Thomas M. Plucinak, Kempton M. Horken, and Donald P. Weeks

University of Nebraska-Lincoln, Biochemistry Dept., 1901 Vine St., Lincoln, NE 68588

The Carbon Concentrating Mechanism (CCM) is the method by which the aquatic microbe C. reinhardtii copes with the low availability of CO2 in its native environment. Under ambient or limiting CO2 conditions, the CCM is activated. This leads to the formation of a special substructure of the chloroplast called the pyrenoid. The majority of the cell's Rubisco is concentrated within this structure. CO2 is actively transported into the pyrenoid to maximize the carboxylation efficiency of the concentrated Rubisco. When CO2 is ample, the CCM is inactivated, the pyrenoid dissociates and Rubisco diffuses throughout the chloroplast. The constitutively expressed CIA5 protein controls the induction of the CCM machinery at the transcriptional level. The cia5 mutant is unable to induce most CO2-responsive genes under limiting CO2 conditions. Despite this protein's importance with respect to photosynthetic control, little is known about its regulation in vivo. An affinity-tagged version of the CIA5 gene has been used to complement the cia5 mutant. We have purified and initially characterized denatured, affinity-tagged CIA5. CIA5 purified from cells growing under varying CO2 levels will be assayed via mass spectrometry to investigate the nature of any post translational modifications that may influence its function in changing CO2 environments.

e-mail address of presenting author: thomas.plucinak@huskers.unl.edu