Abstract

This dissertation reports the function of Nhx1, an endosomal Na+ (K+)/H+ exchanger, in late endosome – vacuole membrane fusion in the model eukaryote S. cerevisiae. Nhx1 is found on the late endosome (LE) where it is known to play a pivotal role in endocytosis. Specifically, loss-of-function mutations in NHX1 block delivery of internalized surface proteins and newly synthesized proteins to the vacuole, the equivalent of the metazoan lysosome in yeast. Because membrane fusion between late endosomes and vacuoles is the final event necessary for protein delivery, and because Nhx1 binds Gyp6, a Rab-GTPase activating protein that is predicted to regulate membrane fusion, I hypothesized that Nhx1 may play a role in LE – vacuole membrane fusion. Unfortunately, there are no existing assays that directly measure this fusion event. Thus, to test this hypothesis, I first devised and optimized an in vitro LE – vacuole membrane fusion assay, which relies on the assembly of complementary β-lactamase fragments to form an active enzyme upon lumenal content mixing. I then used this biochemical assay to characterize the ions and protein machinery responsible for this fusion event. I then demonstrate that Nhx1 is important for LE-vacuole fusion, but its role in this process is independent of its interaction with Gyp6. Together, these results support a model of LE – vacuole fusion that requires H+-transport by Nhx1 upstream of SNARE mediated bilayer mixing.