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 predominantly 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 24–6) 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. GENERAL SIGNIFICANCE 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.

Lactosylceramide contributes to mitochondrial dysfunction in diabetes

“…In STZ-induced diabetes, the lack of insulin releases suppression of lipolysis in adipocytes ( 67,68 ), resulting in elevated levels of FFA in plasma (69)(70)(71)(72), accompanied by expression of is not in direct equilibrium with the pool of ceramide accessible to NCDase, and that the increase in SM in mitochondrial subpopulations of diabetic hearts refl ects a suppression of SM hydrolysis rather than activation of SM synthesis. In line with this notion, there is no evidence of the SM synthase presence in mitochondria, but a novel mitochondria-associated neutral SMase has been cloned and characterized ( 12,13,83 ).…”

“…Neutral ceramidase (NCDase) ( 9-11 ), a novel neutral SMase ( 12,13 ), CerS ( 14 ), and sphingosine kinase 2 ( 10,15 ), which convert sphingosine to sphingosine-1-phosphate, have been found associated with mitochondria. Mitochondria contain a variety of sphingolipid species and some of their effects on mitochondrial function are well-documented ( 16 ).…”

Inhibitors of the sphingomyelin cycle: Sphingomyelin synthases and sphingomyelinases

“…Murine Ma-nSMase requires Mg++ similarly to other nSMases, and it is activated by anionic phospholipids such as PS and cardiolipin (a mitochondrial lipid). It is associated with the outer mitochondrial membrane but its biological function is still unknown (Rajagopalan et al, 2015).…”

Sphingolipids as Emerging Mediators in Retina Degeneration

“…These enzymes present several isoforms differing in subcellular localization, optimal pH range and cation dependence. A prominent example is neutral SMase; a Mg 2+ -dependent form is localized in the plasma membrane whereas a cation-independent form is found in cytosol (Marchesini and Hannun, 2004); a mitochondrial neutral SMase has also been identified (Wu et al, 2010; Rajagopalan et al, 2015). The acid SMase gene can also generate, through differential trafficking, a cation-independent acid SMase, found in the endosomal-lysosomal compartment and an acid SMase that is secreted extracellularly and is responsible for hydrolyzing SM in the outer leaflet of the plasma membrane in addition to that present in plasma lipoproteins (Jenkins et al, 2009).…”