Protein Phosphorylation in the Archaea

Virginia Polytechnic Institute And State University, Blacksburg VA

Investigators

Abstract

The survival of living organisms and the development of complex life forms depend largely upon the proper coordination and control of the molecular events that take place inside cells. The enzyme-catalyzed interconversion of proteins between physically and functionally distinct phosphorylated and dephosphorylated forms serves as a molecular switch that permits proteins to be turned "on" and "off" as needed. The highly effective nature of the phosphate group as an agent for altering the functional properties of proteins coupled with the relative ease with which the phosphate group can be removed render phosphorylation-dephosphorylation uniquely potent and versatile. Consequently, it has been applied to the modulation of virtually every aspect of cell growth, differentiation, motion, metabolism, and communication. The long-term goal of this project is to uncover the fundamental principles through which this regulatory mechanism operates, by tracing its evolutionary history. This project will exploit the phylogenetic diversity of nature to search for and study vestiges of these early events. A set of primitive prokaryotic organisms, the Archaea, serve to some degree as a set of "living fossils" that preserve within their physical, genetic, and biochemical makeup strong echoes of the organisms of an earlier epoch. This project will explore protein phosphorylation-dephosphorylation events in the extreme acidothermophilic archaeon Sulfolobus solfataricus. The gene for a protein-serine/threonine phosphatase, PP1-arch1, exhibiting 30% identity with the PPP-family of protein phosphatases from eukaryotes, has been cloned. This discovery indicates the existence of a continuous line of development/evolution between the protein phosphatases in the Archaea and those in Eukaryotes. The proposed study will dissect at the molecular level the protein kinase(s) and phosphorylated proteins that conform the archaeal protein phosphorylation network. An Archaeal protein kinase will be purified by a combination of chromatographic and electrophoretic techniques, its amino acid sequence will be determined and the corresponding gene will be cloned. Availability of the cloned gene will allow determination of the complete, DNA-derived amino acid sequence of the enzyme and expression of the protein using recombinant DNA technology. Phosphorylated proteins from Sulfolobus solfataricus will be isolated and characterized.