INCORPORATION OF PROTEINS INTO (XENOPUS) OOCYTES BY PROTEOLIPOSOME MICROINJECTION - FUNCTIONAL-CHARACTERIZATION OF A NOVEL AQUAPORIN

Xenopus laevis oocytes are widely used as an expression system for pla sma membrane proteins, achieved by cytoplasmic microinjection of messe nger RNA, In the present study, we propose an alternative system allow ing functional insertion of exogenous proteins into the plasma membran e of Xenopus oocytes, We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function, The protei ns used in this work were members of the MIP family: the human erythro cyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MI P26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-suc king insect (Cicadella viridis), Proteoliposomes containing either AQP 1, MIP26, or P25 were injected into Xenopus oocytes, The subsequent in sertion of these proteins into the plasma membrane of oocytes was demo nstrated by immunocytochemistry. Oocytes microinjected with either AQP 1 or P25-proteoliposomes exhibited significantly increased osmotic mem brane water permeabilities (P-f 3.16+/-026 and 4.03+/-0.26x10(-3) cm/s econd, respectively) compared to those measured for oocytes injected w ith liposomes alone or with MIP26-proteoliposomes (P-f = 1.39+/-0.07 a nd 1.44+/-0.10x10(-3) cm/second, respectively), These effects were inh ibited by HgCl2 in a reversible manner, Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte pla sma membranes (E(a) = 2.29 and 3.01 kcal/mol, respectively, versus E(a ) = 11.75 kcal/mol for liposome injected oocytes). The properties obse rved here for AQP1 are identical to those widely reported following AQ P1 cRNA expression in oocytes. From the present study, we conclude tha t: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequent ly found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties simila r to those described for the archetype of water channels AQP1, and thu s is a new member of the aquaporin family.