Poster: Respiratory pathways

Abs # 214: The uncoupling protein of Chlamydomonas reinhardtii

Presenter:	Berry-Lowe, Sandra L., slblowe@uccs.edu
Authors	Berry-Lowe, Sandra L. (A)   Dupree, Cecily E. (A)   Newell, Karen M. (A)
Affiliations:	(A): University Of Colorado

Chlamydomonas reinhardtii is a unicellular alga that has been a useful model for many types of studies, including photosynthesis and motility. ATP synthesis in photosynthetic organisms is produced by ATP synthase as a result of proton motive force and light energy. The proton gradient (necessary to drive proton motive force), in both mammalian cells and in some plants, can be dissipated by members of family of molecules known as the uncoupling proteins (UCPs). Another function that has been attributed to uncoupling proteins is protection from free radical damage. We previously reported that a C. reinhardtii mutant (nitrate reductase mutant nit-1-305 cw15) expresses a low level of uncoupling protein as detected by flow cytometrically using an antibody to mouse UCP-2. Furthermore, transformation of this C. reinhardtti strain with the mouse UCP-2 gene fused to C. reinhardtii green fluorescent protein (Entelechon) showed increased "anti-mouse UCP-2 detectable" UCP-2. In the present study, we searched the C. reinhardtii dEST database for putative uncoupling protein sequence homolgies, synthesized appropriate primers, and used PCR to isolate the gene from C. reinhardtii. We verified the sequence by nucleotide sequencing, cloned the gene into the pET32 expression vector (Novagen), and are now using the vector to synthesize enough protein to produce C. reinhardtii specific antibody. We plan to use this antibody to extend and confirm our previous flow cytometric analyses.