DICTYOSTELIUM GA2:CELLULAR LOCALIZATION &PALMITOYLATION

University Of Maine Orono, Orono ME

Investigators

Abstract

DESCRIPTION (from applicant's abstract): The protozoan, Dictyostelium discoideum, provides a powerful system in which to examine fundamental signal transduction processes of the eukaryotic cell. During the initial aggregation stage of its developmental life cycle, Dictyostelium utilizes a G protein signal transduction network very similar to those found in more complex organisms. Preliminary results characterizing the cellular location off G-alpha-2, a G protein alpha-subunit essential to Dictyostelium's developmental life cycle, reveal that G-alpha-2 is located in both the pellet and supernatant fractions of cell lysates. The distribution is roughly equal between the two fractions. The goals of this proposal are to determine the physiological distribution of G-alpha-2 in the cell, what factors are responsible for either its particulate or soluble nature and how might they be regulated. Protein acylation is known to be an important determinant in the cellular location of the alpha-subunits. Dictyostelium G-alpha-2 contains the required amino acid sequence for both myristoylation and palmitoylation. Changing these required amino acids by site-directed mutagenesis blocks the developmental life cycle of Dictyostelium. Cells expressing the G-alpha-2-C4A mutant are no longer able to aggregate, demonstrating that palmitoylation is key to alpha-subunit function. Since considerably less is known about the role of protein palmitoylation, research will focus on G-alpha-2 palmitoylation and its possible role in the cellular and localization and function of G-alpha-2. The investigator will examine beta-gamma binding, receptor coupling, effector activation, and other potential protein-protein interactions. He will also identify and characterize in Dictyostelium the enzymes responsible for protein palmitoylation, protein-palmitoyl acyltransferase and protein-palmitoyl thioesterase, using in vitro enzyme assays. The genes for these enzymes will be identified using restriction enzyme-mediated integration (REMI) mutagenesis coupled to screens designed to isolate mutations in G-alpha-2 function.