title: Critical determinants of mitochondria-associated neutral sphingomyelinase (MA-nSMase) for mitochondrial localization.
abstract: BACKGROUND
  A novel murine mitochondria-associated neutral sphingomyelinase (MA-nSMase) has been recently cloned and partially characterized. The subcellular localization of the enzyme was found to be predominant in mitochondria. In this work, the determinants of mitochondrial localization and its topology were investigated.
  
  
  METHODS
  MA-nSMase mutants lacking consecutive regions and fusion proteins of GFP with truncated MA-nSMase regions were constructed and expressed in MCF-7 cells. Its localization was analyzed using confocal microscopy and sub-cellular fractionation methods. The sub-mitochondrial localization of MA-nSMase was determined using protease protection assay on isolated mitochondria.
  
  
  RESULTS
  The results initially showed that a putative mitochondrial localization signal (MLS), homologous to an MLS in the zebra-fish mitochondrial SMase is not necessary for the mitochondrial localization of the murine MA-nSMase. Evidence is provided to the presence of two regions in MA-nSMase that are sufficient for mitochondrial localization: a signal sequence (amino acids 24-56) that is responsible for the mitochondrial localization and an additional 'signal-anchor' sequence (amino acids 77-99) that anchors the protein to the mitochondrial membrane. This protein is topologically located in the outer mitochondrial membrane where both the C and N-termini remain exposed to the cytosol.
  
  
  CONCLUSIONS
  MA-nSMase is a membrane anchored protein with a MLS and a signal-anchor sequence at its N-terminal to localize it to the outer mitochondrial membrane.
  
  
  CONCLUSIONS
  Mitochondrial sphingolipids have been reported to play a critical role in cellular viability. This study opens a new window to investigate their cellular functions, and to define novel therapeutic targets.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173, Ashley Avenue, Charleston, SC 29425, USA.
authors.forename: Vinodh
authors.initials: V
authors.lastname: Rajagopalan
authors.affiliation: Stony Brook Cancer Center and the Department of Medicine, Stony Brook University, Health Sciences Center, Stony Brook, NY 11794, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Canals
authors.affiliation: Stony Brook Cancer Center and the Department of Physiology and Biophysics, Stony Brook University, Health Sciences Center, Stony Brook, NY 11794, USA.
authors.forename: Chiara
authors.initials: C
authors.lastname: Luberto
authors.affiliation: Stony Brook Cancer Center and the Department of Medicine, Stony Brook University, Health Sciences Center, Stony Brook, NY 11794, USA.
authors.forename: Justin
authors.initials: J
authors.lastname: Snider
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173, Ashley Avenue, Charleston, SC 29425, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Voelkel-Johnson
authors.affiliation: Stony Brook Cancer Center and the Department of Medicine, Stony Brook University, Health Sciences Center, Stony Brook, NY 11794, USA.
authors.forename: Lina M
authors.initials: LM
authors.lastname: Obeid
authors.affiliation: Stony Brook Cancer Center and the Department of Medicine, Stony Brook University, Health Sciences Center, Stony Brook, NY 11794, USA. Electronic address: Yusuf.Hannun@stonybrookmedicine.edu.
authors.forename: Yusuf A
authors.initials: YA
authors.lastname: Hannun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D051336:Mitochondrial Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013108:Sphingomyelin Phosphodiesterase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D013108:Sphingomyelin Phosphodiesterase
keywords: MA-nSMase; MLS; Mitochondrion; Sphingolipid; Sphingomyelinase