Identification of small subunits of mammalian serine palmitoyltransferase that confer distinct acyl-CoA substrate specificities

Abstract

Serine palmitoyltransferase (SPT) catalyzes the first committed step in sphingolipid biosynthesis. In yeast, SPT is composed of a heterodimer of 2 highly-related subunits, Lcb1p and Lcb2p, and a third subunit, Tsc3p, which increases enzyme activity markedly and is required for growth at elevated temperatures. Higher eukaryotic orthologs of Lcb1p and Lcb2p have been identified, but SPT activity is not highly correlated with coexpression of these subunits and no ortholog of Tsc3p has been identified. Here, we report the discovery of 2 proteins, ssSPTa and ssSPTb, which despite sharing no homology with Tsc3p, each substantially enhance the activity of mammalian SPT expressed in either yeast or mammalian cells and therefore define an evolutionarily conserved family of low molecular weight proteins that confer full enzyme activity. The 2 ssSPT isoforms share a conserved hydrophobic central domain predicted to reside in the membrane, and each interacts with both hLCB1 and hLCB2 as assessed by positive split ubiquitin 2-hybrid analysis. The presence of these small subunits, along with 2 hLCB2 isofoms, suggests that there are 4 distinct human SPT isozymes. When each SPT isozyme was expressed in either yeast or CHO LyB cells lacking endogenous SPT activity, characterization of their in vitro enzymatic activities, and long-chain base (LCB) profiling revealed differences in acyl-CoA preference that offer a potential explanation for the observed diversity of LCB seen in mammalian cells.

Fig. 1.

ssSPTa and ssSPTb enhance the growth of yeast cells expressing human and yeast SPT heterodimers. Yeast lcb1Δlcb2Δ mutant cells (TDY8055) expressing hLCB1–hLCB2a (A), hLCB1–hLCB2b (B), or tsc3–2 mutant cells, which lack Tsc3p but contain yeast Lcb1–Lcb2p (C), with empty vector (Top), ssSPTa (Middle), or ssSPTb (Bottom) were transferred to yeast extract/peptone/dextrose plates with or without PHS and incubated at 26 °C or 37 °C.

Fig. 2.

ssSPTa and ssSPTb define a new gene family. (A) The ssSPT proteins are homologous to each other but share no homology with yeast Tsc3p. (B and C) The ssSPTa (B) and ssSPTb (C) subfamilies contain a highly-conserved central domain that extends from just after the first predicted membrane spanning domain (TMD1) through TMD2.

Fig. 3.

ssSPTa stimulates SPT activity in mammalian cells. CHO-LyB cells were transfected with plasmids expressing hLCB1, hLCB2a, hLCB2b, or HA-tagged ssSPTa as indicated. Microsomal protein was prepared from the transfected cells. (A) Expression of each protein was analyzed by immunoblotting using antipeptide antibodies for detecting hLCB1, hLCB2a, and hLCB2b and anti-HA antibodies for detecting HA-ssSPTa. (B) SPT activity was determined as described in Materials and Methods.

Fig. 4.

Interactions between human SPT subunits. (A) Solubilized yeast microsomes prepared from cells expressing hLCB2b–TAP, hLCB1-HA–NubG and NubG-HA–ssSPTa (lanes 5 and 6) or NubG-HA–ssSPTb (lanes 7 and 8) or cells expressing only NubG-HA–ssSPTa (lanes 1 and 2) or NubG-HA–ssSPTb (lanes 3 and 4) were incubated with IgG-conjugated Sepharose, and unbound (U) and bound (B) proteins were detected by immunoblotting. The smaller of the 2 hLCB1-HA–NubG bands seen in the unbound lanes appears to be a proteolytic fragment not present in the holoenzyme. (B) Split-ubiquitin 2-hybrid interactions between hLCB1 and the other SPT subunits were identified by using hLCB1–Cub and C-terminally Nub-tagged hLCB2 isoforms or N-terminally Nub-tagged ssSPT isoforms. Interactions between hLCB2 isoforms and ssSPT isoforms were determined by using N-terminally Cub-tagged hLCB2s and N-terminally Nub-tagged ssSPTs in the presence or absence of untagged hLCB1. Potential interactions between hLCB2 isoforms were investigated by using C-terminally Nub-tagged and N-terminally Cub-tagged proteins. Positive interactions were detected by growth on medium lacking histidine and containing 50 mM aminotriazole.

Fig. 5.

Reconstitution of human SPT activity in yeast. Human SPT subunits were expressed in lcb1Δlcb2Δ double mutant yeast cells in various combinations as indicated. Protein expression was verified by immunoblotting as in Fig. 3 (A) and microsomal SPT activity assayed with [³H]serine and palmitoyl-CoA (B).

Fig. 6.

Human SPT isozymes synthesize different LCBs. (A) LCBs were extracted from yeast expressing the hLCB1–hLCB2a heterodimer alone (Inset) or coexpressed with ssSPTa (dotted line) or ssSPTb (solid line) and analyzed by HPLC. Note that cells expressing only the heterodimer (Inset) accumulate significantly less LCBs than those coexpressing either ssSPTa or ssSPTb. (B) SPT activity was measured in microsomes prepared from yeast expressing each of the 4 isozymes using [³H]serine and acyl-CoAs of the indicated (C12–C20) chain lengths.

Fig. 7.

Expression of ssSPTb-containing isozymes in CHO LyB cells increases C20–LCBs. (A) LCBs were extracted from LyB cells expressing each of the 4 human SPT isozymes and levels of C20–SPH and C20–DHS were quantified. (B and C) Microsomes were prepared from LyB cells expressing hLCB1 and hLCB2a with either ssSPTa (B) or ssSPTb (C), and SPT activity was measured by using [³H]serine and acyl-CoAs of the indicated (C12–C22) chain lengths.