SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1–3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.
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Research Article|
June 13 2007
Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?
Thorsten Hornemann;
Thorsten Hornemann
1
1Institute for Clinical Chemistry, University Hospital Zurich, Rämistrasse 100, CH-8091 Zürich, Switzerland
1To whom correspondence should be addressed (email thorsten.hornemann@usz.ch).
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Yu Wei;
Yu Wei
1Institute for Clinical Chemistry, University Hospital Zurich, Rämistrasse 100, CH-8091 Zürich, Switzerland
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Arnold von Eckardstein
Arnold von Eckardstein
1Institute for Clinical Chemistry, University Hospital Zurich, Rämistrasse 100, CH-8091 Zürich, Switzerland
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Publisher: Portland Press Ltd
Received:
January 04 2007
Revision Received:
February 21 2007
Accepted:
March 01 2007
Accepted Manuscript online:
March 01 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2007 Biochemical Society
2007
Biochem J (2007) 405 (1): 157–164.
Article history
Received:
January 04 2007
Revision Received:
February 21 2007
Accepted:
March 01 2007
Accepted Manuscript online:
March 01 2007
Citation
Thorsten Hornemann, Yu Wei, Arnold von Eckardstein; Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?. Biochem J 1 July 2007; 405 (1): 157–164. doi: https://doi.org/10.1042/BJ20070025
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