Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron-microscopy analysis

The MIP (major intrinsic protein) family is a widespread family of membrane proteins exhibiting two major types of channel properties: aquaporins and solute facilitators. In the present study, freeze-fracture electron microsc opy was used to investigate the oligomerization state of two MIP proteins h eterologously expressed in the plasma membrane of Xenopus laevis oocytes: A QPcic, an aquaporin from the insect Cicadella viridis, and GlpF, a glycerol facilitator from Escherichia coli. Swelling assays performed on oocytes 48 and 72 h following cRNA microinjections showed that these proteins were fu nctionally expressed. Particle density determinations indicated that expres sion of proteins is related to an increase in particle density on the P fra cture face of oocyte plasma membranes. Statistical analysis of particle siz es was performed on protoplasmic fracture faces of the plasma membrane of o ocytes expressing AQPcic and GlpF 72 h after cRNA microinjections. Compared to control oocytes, AQPcic-expressing oocytes exhibited a specific populat ion of particles with a mean diameter of 8.7 +/- 0.1 nm. This value is cons istent with the previously reported tetrameric organization of AQPcic, In a ddition, AQPcic particles aggregate and form orthogonal arrays similar to t hose observed in native membranes of C. viridis, consisting of homo-tetrame rs of AQPcic, On the protoplasmic fracture face of oocytes expressing GlpF, the particle density is increased by 4.1-fold and the mean diameter of spe cifically added particles is 5.8 +/- 0.1 nm, This value fits with a monomer of the 28-kDa GlpF protein plus the platinum-carbon layer. These results c learly demonstrate that GlpF is a monomer when functionally expressed in pl asma membranes of Xenopus oocytes and therefore emphasize the key role of t he oligomerization state of MIP proteins with respect to their function. (C ) 1999 Academic Press.