SPTLC1 binds ABCA1 to negatively regulate trafficking and cholesterol efflux activity of the transporter.
ABCA1 transport of cholesterol and phospholipids to nascent HDL particles plays a central role in lipoprotein metabolism and macrophage cholesterol homeostasis. ABCA1 activity is regulated both at the transcriptional level and at the post-translational level. To explore mechanisms involved in the post-translational regulation of the transporter, we have used affinity purification and mass spectrometry to identify proteins that bind ABCA1 and influence its activity. Previously, we demonstrated that an interaction between beta1-syntrophin stimulated ABCA1 activity, at least in part, be slowing the degradation of the transporter. This work demonstrates that one subunit of the serine palmitoyltransferase enzyme, SPTLC1, but not subunit 2 (SPTLC2), is copurified with ABCA1 and negatively regulates its function. In human THP-I macrophages and in mouse liver, the ABCA1-SPTLC1 complex was detected by co-immunoprecipitation, demonstrating that the interaction occurs in cellular settings where ABCA1 activity is critical for HDL genesis. Pharmacologic inhibition of SPTLC1 with myriocin, which resulted in the disruption of the SPTLC1-ABCA1 complex, and siRNA knockdown of SPTLC1 expression both stimulated ABCA1 efflux by nearly 60% ( p < 0.05). In contrast, dominant-negative mutants of SPTLC1 inhibited ABCA1 efflux, indicating that a reduced level of sphingomyelin synthesis could not explain the effect of myriocin on ABCA1 activity. In 293 cells, the SPTLC1 inhibition of ABCA1 activity led to the blockade of the exit of ABCA1 from the endoplasmic reticulum. In contrast, myriocin treatment of macrophages increased the level of cell surface ABCA1. In composite, these results indicate that the physical interaction of ABCA1 and SPTLC1 results in reduction of ABCA1 activity and that inhibition of this interaction produces enhanced cholesterol efflux.
Tamehiro
Norimasa
N
Lipid Metabolism Unit and Center for Computational and Integrative Biology, Massachusetts General Hospital, Harvard Medical School, 185 Cambridge Street, Boston, Massachusetts 02114, USA.
Zhou
Suiping
S
Okuhira
Keiichiro
K
Benita
Yair
Y
Brown
Cari E
CE
Zhuang
Debbie Z
DZ
Latz
Eicke
E
Hornemann
Thorsten
T
von Eckardstein
Arnold
A
Xavier
Ramnik J
RJ
Freeman
Mason W
MW
Fitzgerald
Michael L
ML
D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016632:Apolipoprotein A-I / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism*; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051102:Serine C-Palmitoyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*
C577836:ABCA1 protein, human; D064286:ATP Binding Cassette Transporter 1; D018528:ATP-Binding Cassette Transporters; D016632:Apolipoprotein A-I; D005229:Fatty Acids, Monounsaturated; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D002784:Cholesterol; C487784:SPTLC1 protein, human; C495089:SPTLC2 protein, human; D051102:Serine C-Palmitoyltransferase; C495088:Sptlc1 protein, mouse; C001996:thermozymocidin