SPTLC1 Binds ABCA1 to Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

SPTLC1 Binds ABCA1 to Negatively Regulate Trafficking and Cholesterol Efflux Activity of the Transporter

Authors: Norimasa Tamehiro, Suiping Zhou, Keiichiro Okuhira, Yair Benita, Cari E. Brown, Debbie Z. Zhuang, Eicke Latz, Thorsten Hornemann, Arnold von Eckardstein, Ramnik J. Xavier, Mason W. Freeman, and Michael L. Fitzgerald

Source: Biochemistry, Volume: 47, Issue: 23, Pages: 6138-6147, Published: 2008

Address: http://apps.isiknowledge.com/full_record.do?product=UA&search_mode=GeneralSearch&qid=1&SID=3EdcFJ@AmofLj7loEAi&page=1&doc=1&colname=WOS

Summary:
This paper presents continuing research on the protein-protein interactions between a cholesterol and lipid tranporter, ATP-binding cassette transporter ABCA1, and a subunit of the serine palmitoyltransferase (SPT) holoenzyme, SPTLC1. The authors found that SPTLC1 blocks exit of ABCA1 from the endoplasmic reticulum and down-regulates ABCA1 efflux activity. Furthermore, this regulatory activity is independent of the enzymatic activity of the SPT holoenzyme.

Prior research from the same group showed that ABCA1 contained a C-terminus motif that conformed to the PDZ class I binding motif. By comparing the proteins that bound to wild-type ABCA1 against those that bound to ABCA1 lacking a functional PDZ binding motif, the group were able to identify several proteins that utilize the PDZ motif to interact with ABCA1. SPTLC1 was one of the proteins identified.

In this paper, the authors studied the interactions of SPTLC1 with ABCA1 in more detail.

First, co-immunoprecipitations were conducted with ABCA1. These experiments assume that if a target protein is captured via antibodies, then proteins associated with that target protein will also be pulled down. This paper found that SPTLC1 and ABCA1 form a complex in human macrophages and liver cells in physiologic settings.

Second, the authors studied the functional significance of this complex by inhibiting SPTLC1. This inhibition was enforced either at the translational level via siRNA knockdown of SPTLC1 mRNA or at the post-translational level via the chemical myriocin. In both cases of reduced SPTLC1 activity, ABCA1 cholesterol efflux to apoA-I was increased. Further testing using myriocin suggested that it was specifically the SPTLC1-ABCA1 complex that down-regulated ABCA1 activity.

Third, expression of two different dominant-negative SPTLC1 mutants (that do not have functional enzymatic activity) still resulted in ABCA1 down-regulation, suggesting that enzymatic activity was not required for negative regulation. Furthermore, expressing SPTLC1 (wild-type and dominant negative) with SPTLC2 to form the SPT holoenzyme still resulted in down-regulation of ABCA1. Lastly, to study the physical distribution of ABCA1 and SPTLC1, confocal microscopy was used. Cells containing ABCA1-GFP and SPTLC1 were compared to cells transfected with only ABCA1-GFP. This experiment revealed that SPTLC1 reduced cell-surface distribution of ABCA1, and ABCA1 tended to localize in the ER. Addition of myriocin disrupted the SPTLC1-ABCA1 complex and resulted in greater surface concentrations of ABCA1. Further experimentation suggested that SPTLC1 negatively regulated cell surface ABCA1 levels but did not affect total ABCA1 protein levels.

Lastly, to study the physical distribution of ABCA1 and SPTLC1, confocal microscopy was used. Cells containing ABCA1-GFP and SPTLC1 were compared to cells transfected with only ABCA1-GFP. This experiment revealed that SPTLC1 reduced cell-surface distribution of ABCA1, and ABCA1 tended to localize in the ER. Addition of myriocin disrupted the SPTLC1-ABCA1 complex and resulted in greater surface concentrations of ABCA1. Further experimentation suggested that SPTLC1 negatively regulated cell surface ABCA1 levels but did not affect total ABCA1 protein levels.

Significance:
ABCA1 is an important transporter of cholesterol to the extracellular apolipoprotein A-I (apoA-I), resulting in the formation of high density lipoprotein (HDL). In patients with Tangier’s disease, a defect in ABCA1 results in a severe reduction of HDL; this leads to atherosclerosis. On the other hand, the SPT holoenzyme is involved in the de novo synthesis of sphingolipids, which become incorporated into specialized lipid rafts and membrane bilayers. Consequently, SPT has been shown to have a positive correlation with atherosclerosis. Studying the effects of interactions between these two proteins could give us more insight into methods of suppressing atherosclerosis.