Le Cahérec F, Bron P, Verbavatz J M, Garret A, Morel G, Cavalier A, Bonnec G, Thomas D, Gouranton J, Hubert J F
URA CNRS 256, Université de Rennes I, France.
J Cell Sci. 1996 Jun;109 ( Pt 6):1285-95. doi: 10.1242/jcs.109.6.1285.
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.
非洲爪蟾卵母细胞被广泛用作质膜蛋白的表达系统，通过将信使核糖核酸胞质显微注射来实现。在本研究中，我们提出了一种替代系统，该系统能使外源蛋白功能性地插入非洲爪蟾卵母细胞的质膜。我们将蛋白脂质体悬浮液显微注射到细胞质中，然后分析膜蛋白功能。本研究中使用的蛋白是MIP家族的成员：人类红细胞水通道水通道蛋白1（AQP1）、牛眼晶状体的主要内在蛋白（MIP26）以及来自同翅目吸汁昆虫（绿蚱蝉）消化道中一种水转运复合体的25 kDa多肽（P25）。将含有AQP1、MIP26或P25的蛋白脂质体注射到非洲爪蟾卵母细胞中。通过免疫细胞化学证明了这些蛋白随后插入到卵母细胞质膜中。与仅注射脂质体或注射MIP26 - 蛋白脂质体的卵母细胞相比，显微注射AQP1或P25 - 蛋白脂质体的卵母细胞表现出显著增加的渗透膜水通透性（Pf分别为3.16±0.26和4.03±0.26×10⁻³ cm/秒）（Pf分别为1.39±0.07和1.44±0.10×10⁻³ cm/秒）。这些效应被HgCl₂以可逆方式抑制。当AQP1或P25存在于卵母细胞质膜中时，水转运的阿累尼乌斯活化能较低（Ea分别为2.29和3.01 kcal/mol，而注射脂质体的卵母细胞Ea为11.75 kcal/mol）。这里观察到的AQP1的特性与在卵母细胞中表达AQP1 cRNA后广泛报道的特性相同。从本研究中，我们得出以下结论：（1）掺入脂质体并显微注射到非洲爪蟾卵母细胞细胞质中的外源质膜蛋白随后以功能状态存在于卵母细胞的质膜中；（2）在这个系统中，在绿蚱蝉消化道中发现的MIP家族的P25多肽表现出与水通道原型AQP1描述的特性相似的特性，因此是水通道蛋白家族的一个新成员。
