Coimmunoprecipitation (co-IP) Analysis for Protein-Protein Interactions in the Neurons of the Cerebral Ganglia of the Land Snails of the Genus Polymita During Aestivation

Abstract

Aestivation is a survival strategy used by many invertebrates, e.g., the Cuban land snails of the genus Polymita, to endure arid and very dry environmental conditions. Key features of aestivation include strong metabolic rate suppression, strategies to retain body water, altered nitrogen metabolism, and mechanisms to preserve and stabilize organs, cells, synaptic plasticity, and macromolecules over many months of dormancy. Translational control (the movement of the ribosome along the mRNA) and therefore of the protein synthesis is an important step to achieving both a hypometabolic state and a reorganization of multiple signaling pathways to optimize long-term viability during aestivation of land snails. In these processes, the translation initiation complex (eIF4F) and the elongation factor-2 (eEF2) play a fundamental role as regulatory complexes of the translational and elongation step. The eIF4F and eEF2 consist of multiprotein complexes controlled by phosphorylation and protein-protein interaction mechanisms. Here we describe a coimmunoprecipitation assay to study the dynamic changes that take place in the AE-BP1/eIF4E and eEF2/p53 multiprotein complexes during the cell protein synthesis (cap-dependent translation) in the nervous system of Polymita during the dry and raining seasonal periods in the eastern part of Cuba (Guantanamo Province). Briefly, ganglion dissected from the nervous system is homogenized. This preparation is used to carry out coimmunoprecipitation of the AE-BP1/eIF4E and eEF2/p53 multiprotein complexes, followed by SDS-PAGE and Western blot analysis.