title: Endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44 functions independently and downstream of multivesicular body formation.
abstract: The multivesicular body (MVB) is an endosomal intermediate containing intralumenal vesicles destined for membrane protein degradation in the lysosome. In Saccharomyces cerevisiae, the MVB pathway is composed of 17 evolutionarily conserved ESCRT (endosomal sorting complex required for transport) genes grouped by their vacuole protein sorting Class E mutant phenotypes. Only one integral membrane protein, the endosomal Na+ (K+)/H+ exchanger Nhx1/Vps44, has been assigned to this class, but its role in the MVB pathway has not been directly tested. Herein, we first evaluated the link between Nhx1 and the ESCRT proteins and then used an unbiased phenomics approach to probe the cellular role of Nhx1. Select ESCRT mutants (vps36Δ, vps20Δ, snf7Δ, and bro1Δ) with defects in cargo packaging and intralumenal vesicle formation shared multiple growth phenotypes with nhx1Δ. However, analysis of cellular trafficking and ultrastructural examination by electron microscopy revealed that nhx1Δ cells retain the ability to sort cargo into intralumenal vesicles. In addition, we excluded a role for Nhx1 in Snf7/Bro1-mediated cargo deubiquitylation and Rim101 response to pH stress. Genetic epistasis experiments provided evidence that NHX1 and ESCRT genes function in parallel. A genome-wide screen for single gene deletion mutants that phenocopy nhx1Δ yielded a limited gene set enriched for endosome fusion function, including Rab signaling and actin cytoskeleton reorganization. In light of these findings and the absence of the so-called Class E compartment in nhx1Δ, we eliminated a requirement for Nhx1 in MVB formation and suggest an alternative post-ESCRT role in endosomal membrane fusion.
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Laura M
authors: LM
authors: Kallay
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205; Department of Biology, Concordia University, Montréal, Quebec H3G 1M8, Canada.
authors: Christopher L
authors: CL
authors: Brett
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Deepali N
authors: DN
authors: Tukaye
authors: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309.
authors: Megan A
authors: MA
authors: Wemmer
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Anthony
authors: A
authors: Chyou
authors: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309.
authors: Greg
authors: G
authors: Odorizzi
authors: Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Electronic address: rrao1@jhmi.edu.
authors: Rajini
authors: R
authors: Rao
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods; D008954:Models, Biological; D008957:Models, Genetic; D057146:Multivesicular Bodies / Q000378:metabolism; D009154:Mutation; D010641:Phenotype; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics* / Q000502:physiology*
chemical_list: C492183:NHX1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers