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 plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 x 10(-3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 x 10(-3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently 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 similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.
Le Cahérec
F
F
URA CNRS 256, Université de Rennes I, France.
Bron
P
P
Verbavatz
J M
JM
Garret
A
A
Morel
G
G
Cavalier
A
A
Bonnec
G
G
Thomas
D
D
Gouranton
J
J
Hubert
J F
JF
D000818:Animals; D051398:Aquaporin 1; D020346:Aquaporins; D001789:Blood Group Antigens; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002463:Cell Membrane Permeability; D000090266:Chromobox Protein Homolog 5; D003593:Cytoplasm / Q000378:metabolism; D005136:Eye Proteins / Q000008:administration & dosage / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007313:Insecta; D007473:Ion Channels / Q000008:administration & dosage / Q000378:metabolism; D008562:Membrane Glycoproteins; D008845:Microinjections; D016253:Microscopy, Immunoelectron; D009865:Oocytes / Q000378:metabolism* / Q000648:ultrastructure; D011506:Proteins / Q000008:administration & dosage* / Q000378:metabolism*; D011510:Proteolipids; D014867:Water / Q000378:metabolism; D014982:Xenopus laevis
C496020:AQP1 protein, human; D020346:Aquaporins; D001789:Blood Group Antigens; C000715594:CBX5 protein, human; D005136:Eye Proteins; D007473:Ion Channels; D008562:Membrane Glycoproteins; D011506:Proteins; D011510:Proteolipids; C030328:aquaporin 0; C020485:proteoliposomes; D014867:Water; D000090266:Chromobox Protein Homolog 5; D051398:Aquaporin 1