LOW-MOLECULAR-MASS GTP-BINDING PROTEINS IN CHLAMYDOMONAS-REINHARDTII WILD-TYPE AND A WALL-LESS STRAIN - CHARACTERIZATION AND COMPARISON WITH GTP-BINDING PROTEINS OF DUNALIELLA-SALINA

Low molecular weight, ras-related GTP-binding proteins have been impli cated in several key steps of intracellular vesicle trafficking by yea st and animal cells. The presence of these proteins in plants has also been reported, but much less information is available regarding their function. We have recently presented evidence for the participation o f small GTP-binding proteins in vesicle trafficking by two green algae , Dunaliella salina and Chlamydomonas reinhardtii. Although these two algal species generally resembled each other with respect to their sma ll GTP-binding protein complement, certain dramatic differences were n oted. In this report we characterize and quantify the major small GTP- binding proteins of C. reinhardtii wild type (2137) and a wall-less mu tant (cw15) and compare them with the equivalent proteins of D. salina . Both C. reinhardtii strains contained a characteristic GTP-binding p rotein pattern featuring 21 kDa, 27.5 kDa and 28 kDa components. When grown in minimal media with CO2 bubbling, the 28 kDa protein was the m ajor component, as it was in D. salina. However, when CO2 was replaced by acetate as the carbon source for C. reinhardtii culture, the 27.5 kDa protein predominated. The relative GTP-binding protein content of cell fractions from the C. reinhardtii strains was quantified by bindi ng [P-32]GTP to the proteins on nitrocellulose blots of LDS-PAGE gels. The validity of quantifying GTP-binding protein content in the differ ent species by this means was verified by quantitative immunoblotting. On a per mg total protein basis, fractions from both C. reinhardtii s trains generally had 2-4 times more GTP-binding protein than equivalen t D. salina fractions. The cw15 chloroplast fraction and the wild type cytosolic fraction were exceptional in having 12-13 times more GTP-bi nding protein than their counterparts in D. salina. The intracellular distribution of these GTP-binding proteins among fractions involved in protein trafficking differed among the three algal strains in a way t hat might reflect differences in secretory activity. Because of the re lative abundance of small GTP-binding proteins in C. reinhardtii, this cell is a superior system for the preparation of the proteins and the ir antibodies and for generating probes for their genes. These tools s hould be applicable not only in C. reinhardtii itself but in the close ly related D. salina, where an important role of GTP-binding proteins in intracellular protein trafficking and cell volume control has been proposed.