Observation of membrane proteins in situ: AQPcic, the insect aquaporin example

Aquaporins are water-selective channels widely distributed in prokaryotes, plants, and animals. Looking for the presence of a water channel in the filter chamber (FC) of a homopteran insect (Cicadella viridis), we conducted an electron microscopic study. On thin sections, FC displays thin epithelia with developed basal membrane folds (BMFs). Freeze fracture performed on FC shows an amazing network of intramembrane particles. Epithelial cell membranes were purified and observed by negative staining for control purity. Membrane solubilisation followed by PAGE showed that a 25-kDa polypeptide (P25) is the major protein constituent. Using a specific antibody, we located P25 on thin sections on the microvilli and on BMFs of the epithelial cells. Immunogold localisation of P25 on negatively stained membranes and examination of Pt/C shadowed membranes demonstrated that P25 has an asymmetric insertion within the membrane. cDNA cloning and heterologous expression confirmed that P25 is an aquaporin; thus, we called it AQPcic. The native state of crystallisation of this aquaporin in the membrane appeared to be unique and favourable for a structural investigation by negative staining, cryo-electron microscopy, and image processing. We demonstrated that, in the native membrane, AQPcic is a homotetramer forming a regular two-dimensional array.