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 Xenopus oocytes, we have monitored changes in the surface expression of sodium pumps, the surface area of the oocyte and the uptake of the fluid-phase marker inulin. Xenopus oocytes possess intracellular sodium pumps that are continuously exchanged for surface sodium pumps by constitutive endo- and exocytosis. Injection of Clostridium botulinum C3 exoenzyme, which inactivates Rho by ADP-ribosylation, induced a redistribution of virtually all intracellular sodium pumps to the plasma membrane and increased the surface area of the oocytes. The identical effects were caused by injection of ADP-ribosylated recombinant RhoA into oocytes. The C3 exoenzyme acts by blocking constitutive endocytosis in oocytes, as determined using a mAb to the beta 1 subunit of the mouse sodium pump as a reporter molecule and oocytes expressing heterologous sodium pumps. In contrast, an increase in endocytosis and a decrease in the surface area was induced by injection of recombinant Val14-RhoA protein or Val14-rhoA cRNA. PMA stimulated sodium pump endocytosis, an effect that was blocked by a specific inhibitor of protein kinase C (Gö 16) or by ADP-ribosylation of Rho by C3. Similarly, the phorbol ester-induced increase in fluid-phase endocytosis in oocytes was inhibited by Gö 16, C3 transferase, or by injection of ADP-ribosylated RhoA. In contrast to C3 transferase, C. botulinum C2 transferase, which ADP-ribosylates actin, had no effect on sodium pump endocytosis or PMA-stimulated fluid-phase endocytosis. The data suggests that RhoA is an essential component of a presumably clathrin-independent endocytic pathway in Xenopus oocytes which can be regulated by protein kinase C.
Schmalzing
G
G
Max-Planck-Institut für Biophysik, Frankfurt, Germany.
Richter
H P
HP
Hansen
A
A
Schwarz
W
W
Just
I
I
Aktories
K
K
D036002:ADP Ribose Transferases / Q000378:metabolism; D000818:Animals; D001905:Botulinum Toxins; D004705:Endocytosis / Q000187:drug effects; D005260:Female; D019204:GTP-Binding Proteins / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000502:physiology; D014982:Xenopus laevis / Q000502:physiology*; D020742:rhoA GTP-Binding Protein
D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020742:rhoA GTP-Binding Protein; D000254:Sodium-Potassium-Exchanging ATPase