The small domain of cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 modulates the apparent molecular mass and decreases the accumulation of cytochrome f in the mesophile Chlamydomonas reinhardtii.
Cytochrome f from the psychrophile Chlamydomonas raudensis UWO 241 has a lower thermostability of its c-type heme and an apparent molecular mass that is 7 kDa lower than that of the model mesophilic green alga Chlamydomonas reinhardtii. We combined chloroplast transformation, site-directed mutagensis, and the creation of chimeric fusion constructs to assess the contribution of specific domains and (or) amino acids residues to the structure, stability, and accumulation of cytochrome f, as well as its function in photosynthetic intersystem electron transport. We demonstrate that differences in the amino acid sequence of the small domain and specific charged amino acids in the large domain of cytochrome f alter the physical properties of this protein but do not affect either the thermostability of the c-type heme, the apparent half-life of cytochrome f in the presence of the chloroplastic protein synthesis inhibitor chloramphenicol, or the capacity for photosynthetic intersystem electron transport, measured as e-/P700. However, pulse-labeling with [14C]acetate, combined with immunoblotting, indicated that the negative autoregulation of cytochrome f accumulation observed in mesophilic C. reinhardtii transformed with chimeric constructs from the psychrophile was likely the result of the defective association of the chimeric forms of cytochrome f with the other subunits of the cytochrome b6/f complex native to the C. reinhardtii wild type. These results are discussed in terms of the unique fatty acid composition of the thylakoid membranes of C. raudensis UWO 241 adapted to cold environments.
Gudynaite-Savitch
Loreta
L
Department of Biology and The Biotron, University of Western Ontario, London, ON N6A 5B7, Canada. loretag@iogen.ca
Loiselay
Christelle
C
Savitch
Leonid V
LV
Simmonds
John
J
Kohalmi
Susanne E
SE
Choquet
Yves
Y
Hüner
Norman P A
NP
D000595:Amino Acid Sequence; D000818:Animals; D002696:Chlamydomonas / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D045348:Cytochromes f / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
D011993:Recombinant Fusion Proteins; D045348:Cytochromes f