DISSECTING THE ROLE OF YEAST RIM8 IN THEINTERNALIZATION OF THE PLASMA MEMBRANE PROTON PUMPPMA1

Abstract

The vacuolar A-TPase (V-ATPase) is a proton pump that is found ubiquitiously throughout the cells. It uses the hydrolysis of ATP to transport protons across membranes, thereby maintaining homeostatic pH. pH control in the cells of an organism is vital, a disturbance in cellular pH may be lethal. The maintenance of homeostatic pH within the cell appears to be a result of the interplay between V-ATPasesandproton exporters. In yeast and plants, the major proton exporter isthe plasma membrane proton exporter, Pma1. Pma1 is the transporter that is primarily involved in themaintenance of cytosolic pH. In cells in which the function of V-ATPase has been compromised (vma mutants) Pma1 is partially mislocalized.It is known thatmembrane transporterslacking the PY motifare endocytosed via the action of an Arrestin Related Trafficking (ART) protein, which translocates an E3 ligase into close proximity with the transporter, so as to allow for the ubiquitination of the transporter. Rim 8 is the ART protein (adaptor) that has been linked to the endocytosis of Pma1, along with E3 Ubiquitin ligase Rsp5. It is of interest to this project that Rim8 is well studied in its role as an adaptor in the alkaline ambient pH pathway. We thus propose that there may be crosstalk between the ambient pH pathway and the pathway that leads to the internalization of Pma1. Therefore, in this body of work we seek to find other players that may be involved in the Pma1 pathway, as well as to elucidate theareas of interaction between Rim8 and Pma1. Ourfinal goal isbringing a better understanding ofthe pathway that leads to the endocytosis of Pma1. To answer the question posed in this work we monitored the growth phenotype and the localization of Pma1 indouble mutants lacking both V-ATPase subunits and key players in the ambient pH pathway. In addition, we looked to see which cytosolically exposed terminal of Pma1 may be involved in theinteraction with Rim8. In yet another experiment, we mutatedRim8 so as to find which areas of the adaptor werevital for Pma1 internalization.Our results showed that other players tested in the Rim pathway(vma2∆rim20∆and vma2∆vps23∆)werenot required for Pma1 internalization. In addition we observed that mutations in Rim8 that compromise its function in the Rim pathway still allow Pma1 internalization, even though they show synthetic growth phenotypes with vma2∆ mutations. Two-hybrid assay could not detect thesites of interaction between Rim8 and Pma1 and newstrategies will be employed to determine these sites. Changes in electrophoretic mobility of Rim8 suggested that Rim8 undergoes posttranslational modifications, and showed differences in vma2∆mutants and WT mutants.