The Na⁺(K⁺)/H⁺ exchanger Nhx1 controls multivesicular body-vacuolar lysosome fusion

Abstract

Loss-of-function mutations in human endosomal Na + (K + )/H + Exchangers (NHEs) NHE6 and NHE9 are implicated in neurological disorders including Christianson Syndrome, autism and attention deficit and hyperactivity disorder (ADHD). These mutations disrupt retention of 5 surface receptors within neurons and glial cells by affecting their delivery to lysosomes for degradation. However, the molecular basis of how these endosomal NHEs control endocytic trafficking is unclear. Using Saccharomyces cerevisiae as a model, we conducted cell-free organelle fusion assays to show that transport activity of the orthologous endosomal NHE Nhx1 is important for multivesicular body (MVB)-vacuolar lysosome fusion, the last step of 10 endocytosis required for surface protein degradation. We find that deleting Nhx1 disrupts the fusogenicity of the MVB, not vacuole, by targeting pH-sensitive machinery downstream of the Rab-GTPase Ypt7 needed for SNARE-mediated lipid bilayer merger. All contributing mechanisms are evolutionarily conserved offering new insight into the etiology of human disorders linked to loss of endosomal NHE function.

Distinct features of multivesicular body-lysosome fusion revealed by a new cell-free content-mixing assay

“…As predicted, increasing concentrations of anti-Pep12 antibody preferentially blocked heterotypic fusion, whereas anti-Vam3 antibody preferentially inhibited homotypic fusion. Addition of purified anti-Nyv1 antibody or knocking out NYV1 blocked both 35 fusion events, consistent with the presence of this R-SNARE in both complexes (Figures 3D and S2). Moreover, when we mix mutant and wild type organelles, we find that heterotypic fusion is impaired only when vacuole membranes lack NVY1 ( Figure 3E), confirming that this R-SNARE is donated from the vacuole.…”

“…We next collected fractions containing MVB membranes (9-12) from Pep12-Fos-Gs-ω expressing cells or vacuole membranes (3-4) from CPY50-Jun-Gs-α expressing cells and mixed them together under conditions that promote the homotypic vacuole membrane fusion reaction in vitro (see Jun and Wickner, 2007). Unfortunately these preparations were fusion 35 incompetent ( Figure S1B). Thus, we sought an alternative method to isolate the organelles to optimize our cell-free fusion assay.…”

“…As predicted, deleting any of the three ESCRT components inhibited MVB-vacuole fusion, indicating that blocking any stage of protein sorting into ILVs impairs heterotypic fusion. 35 successive fusion events in the secretory and endocytic pathways for efficient delivery of cargo proteins (Rivera- Molina and Novick, 2009). At the MVB, it has been proposed that inactivation of the Rab5 ortholog Vps21 (responsible for endosomal fusion events that deliver proteins to the MVB) is necessary for activation of Ypt7 to initiate the next trafficking step: MVB-vacuole fusion.…”

“…First, we added GTPγS, a non-hydrolyzable GTP analog that activates all GTPases (including Ypt7) at a concentration (0.2 mM) that supports vacuole-vacuole and MVB-vacuole fusion in vitro (Jun and Wickner, 2007;Mattie et al, 2017;Karim and Brett, 2017), and found 10 that it partially rescued heterotypic fusion defects caused by deleting components of ESCRTs ( Figure 4B). Next, we added 100 nM rVam7 in place of ATP to stimulate fusion, as it bypasses the requirement for Ypt7 (Thorngren et al, 2004), and found that it also partially rescued fusion defects ( Figure 4B and D).…”

“…Vida and Gerhardt, 30 1999), it does not require addition of cytosol indicating that all machinery necessary for fusion co-purifies with organelles facilitating their study. Finally, it offers the advantages of changing the reaction buffer conditions to simulate changes in the "cytoplasm" to better understand how heterotypic fusion is regulated or using protein reagents to overcome issues associated with pleiotropy, allowing study of contributions from gene products predicted to have multiple 35 functions at multiple sites in cells, e.g. Sec17.…”