The Function of Trehalose in Cell Stress

Abstract

Trehalose mobilization in Saccharomyces cerevisiae cells, characterization of the permease located in the plasma membranes which transports trehalose, and cloning and expressing the trehalose transporter gene are the aims of this project. Trehalose mobilization in yeast cells will be approached by establishing the protein phosphatases profile of yeast cells, with special focusing in the protein phosphatase that promotes the dephosphrylation of trehalase. Citoplasmic trehalase is regulated by phosphorylation and is involved with trehalose degradation.Another approach will be the separation of the ADPG from the UDPG-dependent trehalose synthase and the purification of the ADPG-dependent trehalose synthase activity from strains in which the UDPG-dependent trehalose synthase has been deleted. The trehalose transporter expression is regulated by a regulatory gene from one MAL locus. A yeast strain containing the regulatory gene from MAL1 locus and the AGT1 gene which codes for a putative trehalose transporter was constructed. From plasma membrane preparations obtained from these cells a protein which correponds to the AGT1 gene product was isolated. This protein will be characterized, sequenced and reconstituted into phosphlipid vesicles to ascertain its functionality as a trehalose transporter. Cloning and expression of this transporter would allow us to analyze the resulting phenotype in strains specifically deleted in these genes. It would also be possible to control the transport activity by using heterologous promoters, what could lead to the production of large quantities of the transporter protein which is essential for strucutural studies. (AU)