Intracellular translocation of a 28 kDa GTP-binding protein during osmotic shock-induced cell volume regulation in Dunaliella salina

The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identification by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx. 21, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella. The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein. Immunodetection analysis showed that the D. salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein. This antibody was utilized for quantitative GTP-binding protein measurements as described below. Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D. salina 28-kDa protein but also the 29-kDa component. The 30-kDa GTP-binding protein of D. salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins. When D. salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative [32P]GTP binding and [125I]antibody binding on nitrocellulose blots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock. We also examined the behavior of D. salina small GTP-binding proteins following exposure of cells to hyperosmotic shock. Increasing the NaCl concentration from 1.7 M to 3.4 M led within 8 min to a decrease in 28-kDa GTP-binding protein content in ER, GA, plasma membrane and chloroplasts, and a concurrent increase in the cytosol. The pattern of change differed from that seen following hypoosmotic shock, where the plasma membrane and chloroplast fractions, as well as the cytosol gained 28-kDa GTP-binding protein during accelerated membrane vesicle trafficking. It appears that hyperosmotic shock, by interrupting vesicular trafficking, releases the 28-kDa GTP-binding proteins from their membrane associations. Two less abundant GTP-binding proteins were also redistributed following hyperosmotic shock. A 30-kDa component of microsomes decreased in amount, but only after 8 min of shock. And a barely detectable 21-kDa band present in organelle fractions was slowly released into the cytosol, becoming relatively prominent there by 30 min. Our findings suggest a role for small GTP-binding proteins in osmoregulatory volume control by D. salina.