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 pr oteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identificatio n by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thomp son, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presenc e of at least five such proteins, having molecular masses of approx. 2 1, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely i n the endoplasmic reticulum (ER) and in an intermediate density organe lle fraction (GA) containing mainly Golgi vesicles, mitochondria and f lagella. 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 protei n. Immunodetection analysis showed that the D. salina 28-kDa protein c ross-reacted strongly with a polyclonal antibody raised against a Volv ox 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 sou rces, namely, monoclonal antibodies raised against yeast and mouse ypt 1 GTP-binding proteins, cross-reacted not only with the D. salina 28-k Da protein but also the 29-kDa component. The 30-kDa GTP-binding prote in of D. salina did not bind the antibodies mentioned above but did cr oss-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibod ies raised against SEC4, rab1 or rab6 proteins. When D. salina cells w ere subjected to hypoosmotic swelling by abruptly reducing the NaCl co ncentration 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 me mbrane, chloroplast and cytosolic fractions, as determined by quantita tive [P-32]GTP binding and [I-125]antibody binding on nitrocellulose b lots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net m ovement of the 30-kDa GTP-binding protein from either the ER or GA fra ctions 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 pro tein content in ER, GA, plasma membrane and chloroplasts, and a concur rent increase in the cytosol. The pattern of change differed from that seen following hypoosmotic shock, where the plasma membrane and chlor oplast fractions, as well as the cytosol gained 28-kDa GTP-binding pro tein during accelerated membrane vesicle trafficking. It appears that hyperosmotic shock, by interrupting vesicular trafficking, releases th e 28-kDa GTP-binding proteins from their membrane associations. Two le ss abundant GTP-binding proteins were also redistributed following hyp erosmotic shock. A 30-kDa component of microsomes decreased in amount, but only after 8 min of shock. And a barely detectable 21-kDa band pr esent in organelle fractions was slowly released into the cytosol, bec oming relatively prominent there by 30 min. Our findings suggest a rol e for small GTP-binding proteins in osmoregulatory volume control by D . salina.