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Analysis of membrane topology of neutral sphingomyelinase 2
Author(s) -
Tani Motohiro,
Hannun Yusuf A.
Publication year - 2007
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2007.02.046
Subject(s) - membrane , sphingomyelin , cytosol , membrane topology , ceramide , microbiology and biotechnology , mutant , membrane protein , chemistry , cell membrane , ferm domain , enzyme , biophysics , biochemistry , biology , topology (electrical circuits) , gene , integral membrane protein , apoptosis , mathematics , combinatorics
Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH 2 ‐terminus, plays an important role in ceramide‐mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double‐tagged nSMase2 at both the NH 2 and COOH termini, was overexpressed in MCF‐7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein‐fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.