The Sodium/Proton Exchanger Nhx1p Is Required for Endosomal Protein Trafficking in the YeastSaccharomyces cerevisiae

Abstract

We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell. nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.

The Endosomal Na+/H+ Exchanger Contributes to Multivesicular Body Formation by Regulating the Recruitment of ESCRT-0 Vps27p to the Endosomal Membrane

“…4 Because the organellar lumen is maintained at an acidic pH by alternative mechanisms, even in vma2⌬ cells lacking V-ATPase activity (50,51), our data provide no evidence for the requirement of an acidic endosomal lumen in MVB formation. However, this result suggests that Nhx1p can function independently of V-ATPase activity, at least in MVB formation, which is consistent with the results of carboxypeptidase Y secretion in vma2⌬ or nhx1⌬ cells, as reported previously (30). Furthermore, this finding supports the idea that Nhx1p contributes to MVB formation by FIGURE 9.…”

“…Previous genetic and morphological studies of yeast mutant cells revealed that Nhx1p is a class E protein (2,3,30); however, the functional role of Nhx1p in MVB formation has not been reported because no direct assay system for MVB formation was available. Here, we established an in vitro biochemical assay that monitors the formation of internal vesicles in the MVB of yeast cells (Figs.…”

“…Negatively charged residues are putatively involved in the ion-transport activity of Nhx1p (30). To confirm that Nhx1p activity is required for the correct delivery of GFP-Cps1p into the vacuole lumen, the intracellular localization of GFP-Cps1p was analyzed in nhx1⌬ cells transformed with plasmids carrying either wild-type NHX1 or mutant Nhx1p (Nhx1p-E225Q/ D230N), in which two negatively charged residues, Glu-225 and Asp-230, were replaced by Gln and Asn.…”

“…When grown in medium with extremely low pH (Ͻ4.0), NHX1-disrupted (nhx1⌬) cells show defects in cell growth and excess acidification of the vacuolar lumen compared with wild-type cells (28,29), suggesting that Nhx1p also contributes to pH homeostasis of organelles. In addition, loss of NHX1 causes a VPS phenotype that is characterized by the missorting of vacuolar carboxypeptidase Y to the extracellular space, improper transport of vacuolar proteins, and enlargement of the late endosomes (class E compartment) (29,30). Accordingly, the NHX1 gene is allelic to VPS44, which was classified as belonging to the class E VPS genes (30).…”

“…In addition, loss of NHX1 causes a VPS phenotype that is characterized by the missorting of vacuolar carboxypeptidase Y to the extracellular space, improper transport of vacuolar proteins, and enlargement of the late endosomes (class E compartment) (29,30). Accordingly, the NHX1 gene is allelic to VPS44, which was classified as belonging to the class E VPS genes (30).…”