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.

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.

Keywords: MA-nSMase; MLS; Mitochondrion; Sphingolipid; Sphingomyelinase.

Fig. 1

Fig. 1A. Sequence alignment of SMPD 5 (MA-nSMase) with SMPD 3 (nSMase 2). Alignment of the deduced amino acid sequences of human (NP001182466.1), mouse (XP002692632.1), bovine (XP003120137.2) and zebrafish (NP001071083.1) SMPD5; and human (NP001116222.1), mouse (XP005256088.1), bovine (NP001179292.1), and zebrafish (AAH43077.1) SMPD3. The sequences were aligned by Clustal omega program. Identical residues in all the three sequences are indicated by bold characters. The mitochondrial localization signal (MLS) of the zebrafish mitochondrial SMase that spans 1–35aa which is homologous to residues 24–56aa of MA-nSMase highlighted in red, predicted transmembrane domain (TMD) sequence spanning between 77–99aa in MA-nSMase is highlighted in yellow and the catalytic domain that spans 129–483 in the mouse MA-nSMase, predicted based on nSMase 2 sequence is highlighted in pink. Fig. 1B, Schematic representation of the MLS, TMD and catalytic domain of MA-nSMase. C, Truncated mutant constructs and its co-localization with mitochondrial marker (Tom 20). Table 1 showing the summary of the results of truncation mutant constructs.

Fig. 1

Fig. 1A. Sequence alignment of SMPD 5 (MA-nSMase) with SMPD 3 (nSMase 2). Alignment of the deduced amino acid sequences of human (NP001182466.1), mouse (XP002692632.1), bovine (XP003120137.2) and zebrafish (NP001071083.1) SMPD5; and human (NP001116222.1), mouse (XP005256088.1), bovine (NP001179292.1), and zebrafish (AAH43077.1) SMPD3. The sequences were aligned by Clustal omega program. Identical residues in all the three sequences are indicated by bold characters. The mitochondrial localization signal (MLS) of the zebrafish mitochondrial SMase that spans 1–35aa which is homologous to residues 24–56aa of MA-nSMase highlighted in red, predicted transmembrane domain (TMD) sequence spanning between 77–99aa in MA-nSMase is highlighted in yellow and the catalytic domain that spans 129–483 in the mouse MA-nSMase, predicted based on nSMase 2 sequence is highlighted in pink. Fig. 1B, Schematic representation of the MLS, TMD and catalytic domain of MA-nSMase. C, Truncated mutant constructs and its co-localization with mitochondrial marker (Tom 20). Table 1 showing the summary of the results of truncation mutant constructs.

Fig. 2. Co-localization and cellular fractionation of Wild type and deletion of MLS (ΔMLS) MA-nSMase constructs

A and B, MCF-7 transfected with WT (panel A) and MCF-7 transfected with ΔMLS (Δ24–56) (panel B) cells were transfected with MA-nSMase expression vector. After 24 h, the cells were fixed and co-stained with an antibody against V5 (green) for MA-nSMase signal and antibodies against various subcellular markers (red), including Tom20 (mitochondrial markers), calreticulin (ER marker) and then subjected to confocal microscopic observation. The colocalization signals were observed as yellow or orange and quantified using Pearson correlation coefficient (PCC) which measures the pixel-by-pixel covariance in the signal levels of two images. On to the right of WT (panel A), ΔMLS (Δ24–56) (panel B), cellular lysates from corresponding constructs were incubated in isotonic buffer containing 250mM sucrose, disrupted by passage through 25 gauge needle and centrifuged at 1,000 × g for 10 min (indicated as 1KP in the western blot figure) and 10,000 × g for 10 min (indicated as 10 KP in the western blot figure) for collection of the nuclear fraction and the mitochondria-enriched heavy membrane fraction respectively. The supernatant obtained after the 10,000 × g was used for light membrane and cytosolic fraction. The whole cell lysate was used to show the input. Fractions were analyzed by western blotting. WCL: whole cell lysate; 1KP: 1,000g pellet; 10KP: 10,000g pellet; 10KS: 10,000g supernatant.

Fig. 3. Co-localization and cellular fractionation of deletion of TMD (Δ77–99)

A. MCF-7 cells were transfected with an expression construct of deletion of TMD MA-nSMase; co-localization and fractionation were done as described previously. B. Co-localization and cellular fractionation of double deletion of MLS and TMD (Δ24–99): MCF-7 cells were transfected with an expression construct of double deletion of MLS and TMD MA-nSMase; co-localization and fractionation were done as described previously. C. Co-localization and cellular fractionation of deletion of residues 99–119aa flanking the transmembrane domain(Δ99–119): MCF-7 cells were transfected with an expression construct of deletion of residues 99–119aa flanking the transmembrane domain of MA-nSMase; co-localization and fractionation were done as described previously.

Fig. 4. Co-localization of various GFP fusion constructs

MCF-7 cells were transfected with a series of GFP fusion constructs made with aforementioned critical regions such as putative MLS 24–56-GFP, TMD 77–99-GFP, the TMD flanking region 99–119-GFP, and the TMD with its flanking region 77–119-GFP and subjected to confocal visualization as described previously.

Fig. 5. Co-localization of bacterial SMase fusion constructs

MCF-7 cells were transfected with a series of bacterial SMase fusion constructs such as 1–66, 1–128 of MA-nSMase fused to bacterial SMase and Δ24–56 on 1–128 bSMase construct. These constructs were subjected to confocal visualization and differential centrifugation as described previously.

Fig. 5. Co-localization of bacterial SMase fusion constructs

MCF-7 cells were transfected with a series of bacterial SMase fusion constructs such as 1–66, 1–128 of MA-nSMase fused to bacterial SMase and Δ24–56 on 1–128 bSMase construct. These constructs were subjected to confocal visualization and differential centrifugation as described previously.

Fig. 6. Topology of MA-nSMase as demonstrated by the protease protection assay

V5 tags were introduced at C terminal, between residues 184 to 185 and at N terminal. Crude heavy membrane fraction (10,000 ×g pellet) was collected as described previously from MCF-7 cells expressing these constructs. The pellet was suspended in the isotonic buffer and the protein concentration measured using BCA assay (Bio-Rad Laboratories). 5mg/mL of protein were used as an input for the protease protection assay. Proteinase K was added from freshly prepared stocks in water. Unless otherwise indicated, the final proteinase K concentration was 4µg/ml and the detergent Triton X-100 was added at 0.2% (w/v) final concentration. Digestion reactions were performed for 20 min on ice and quenched by addition of PMSF to a final concentration of 2 mM. Finally, the samples were analyzed by western blotting.