Different Behaviours of MIP Proteins in N-Lauroylsarcosine

Abstract

Purification of MIP proteins is of fundamental interest for further reconstitution in artificial lipid bilayers and determination of functional properties or structural analysis by cryoelectron microscopy of 2D-crystals. It has been shown that native or recombinant AQP1 can be purified by a few steps procedure using N-lauroylsarcosine (NLS) as detergent (Smith & Agre, 1991). A first extraction with NLS solubilizes almost all the membranous proteins, except AQP1 which remains in 100,000g pellet. The aquaporin can then be obtained at a good degree of purity by resuspending the NLS insoluble extract in n-octyl glucoside (OG). We successfully used this procedure to purify the insect aquaporin expressed in yeast. This protocol allows the protein to exhibit water channel properties in proteoliposomes and to retain its homotetrameric native state (Lagrée et al., 1998, 1999). In the MIP family, we observed that the E. coli glycerol facilitator, GlpF, is monomeric in the non-denaturing detergents OG (2%, w/v) and Triton X-100 (1%, v/v), while aquaporins are tetrameric. We thus hypothesised the existence of a relationship between the functional specificity and the quaternary structural organisation of MIPs. As mentioned earlier, NLS resistance is a well established physicochemical characteristic of the aquaporins AQP1 and AQPcic. In the present study, we analysed the behaviour in 2% NLS (w/v) of the solute facilitator GlpF and several mutants of AQPcic.