| Interwoven into the fabric of life, pH is sensed and cautiously regulated during all aspects of cellular physiology. In an ongoing project entitled "pHome", I developed in situ probes of cytoplasmic and vacuolar pH to analyze the response of 4,800 yeast deletion mutants to changes in environmental pH. Among the many sensors, regulators and direct contributors to cellular pH identified, herein. I focus on the endosomal Na +(K+)/H+ exchanger, Nhx1. I began to define its role in ion homeostasis by identifying novel salt and acid-sensitive phenotypes in the nhx1 null mutant. Specifically, I showed that Nhx1 serves as a "proton leak" pathway to alkalinize the endosomal lumen and regulate vesicular trafficking. By screening a panel of amiloride derivatives from Aventis Pharmaceuticals, I identified a specific inhibitor of Nhx1. Using this inhibitor and a large-scale phenomics approach, I further defined the role of Nhx1 as mediator of vesicle formation in cellular pathways of endosomal exit, including the multivesicular body pathway responsible for vacuolar mediated protein degradation and retrovirus particle formation (e.g. HIV-1). Detailed phylogenetic analysis established Nhx1 as a founding member of a newly-defined branch of the NHE gene family localized to endomembranes, including human orthologs NHE6, NHE7 and NHE9. Like yeast Nhx1, I showed that human NHE6 localizes to the recycling endosome of mammalian cells. Thus, yeast Nhx1 is a good model for the study of pH-mediated vesicle formation and use of inhibitors specific to human Nhx1 orthologs may be clinically relevant for treatment of retroviral infections, such as HIV. |
