INVOLVEMENT OF THE GTP-BINDING PROTEIN-RHO IN CONSTITUTIVE ENDOCYTOSIS IN XENOPUS-LAEVIS OOCYTES

To study an endocytotic role of the GTP-binding protein RhoA in Xenopu s oocytes, we have monitored changes in the surface expression of sodi um pumps, the surface area of the oocyte and the uptake of the fluid-p hase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitut ive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenz yme, which inactivates Rho by ADP-ribosylation, induced a redistributi on of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects w ere caused by injection of ADP-ribosylated recombinant RhoA into oocyt es. The C3 exoenzyme acts by blocking constitutive endocytosis in oocy tes, as determined using a mAb to the beta 1 subunit of the mouse sodi um pump as a reporter molecule and oocytes expressing heterologous sod ium pumps. In contrast, an increase in endocytosis and a decrease in t he surface area was induced by injection of recombinant Val14-RhoA pro tein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an ef fect that was blocked by a specific inhibitor of protein kinase C (Go 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester- induced increase in fluid-phase endocytosis in oocytes was inhibited b y Go 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In c ontrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribo sylates actin, had no effect on sodium pump endocytosis or PMA-stimula ted fluid-phase endocytosis. The data suggests that RhoA is an essenti al component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.