Topological and Functional Characterization of the ssSPTs, Small Activating Subunits of Serine Palmitoyltransferase

Jeffrey M Harmon,Dagmar Bacikova,Kenneth Gable,Sita D Gupta,Gongshe Han,Nivedita Sengupta,Niranjanakumari Somashekarappa,Teresa M Dunn

doi:10.1074/jbc.m113.451526

Abstract

The topological and functional organization of the two isoforms of the small subunits of human serine palmitoyltransferase (hssSPTs) that activate the catalytic hLCB1/hLCB2 heterodimer was investigated. A variety of experimental approaches placed the N termini of the ssSPTs in the cytosol, their C termini in the lumen, and showed that they contain a single transmembrane domain. Deletion analysis revealed that the ability to activate the heterodimer is contained in a conserved 33-amino acid core domain that has the same membrane topology as the full-length protein. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met(25) in ssSPTa and Val(25) in ssSPTb) was identified which confers specificity for palmitoyl- or stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue also determines which isoform is a better activator of a mutant heterodimer, hLCB1(S331F)/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. This suggests that the role of the ssSPTs is to increase SPT activity without compromising substrate specificity. In addition, the observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered.

Highlights

The ssSPTs activate serine palmitoyltransferase and specify its acyl-CoA selectivity
The ssSPTs behave like integral membrane proteins when coexpressed with hLCB1 and hLCB2a in both yeast (Fig. 2A, upper) and mammalian cells (Fig. 2A, lower); in either case, the proteins are solubilized with detergents, but not by salt, bicarbonate, or urea
To directly address the location of the C terminus, a glycosylation cassette (GC) containing three consensus NX(S/T) glycosylation sites was appended to the C terminus of ssSPTa or ssSPTb and tagged proteins assessed for glycosylation based on their sensitivity to EndoH treatment

Summary

Introduction

The ssSPTs activate serine palmitoyltransferase and specify its acyl-CoA selectivity. In combination with analysis of isoform chimera and site-directed mutagenesis, a single amino acid residue in this core (Met in ssSPTa and Val in ssSPTb) was identified which confers specificity for palmitoylor stearoyl-CoA, respectively, in both yeast and mammalian cells. This same residue determines which isoform is a better activator of a mutant heterodimer, hLCB1S331F/hLCB2a, which has increased basal SPT activity and decreased amino acid substrate selectivity. The observation that the C-terminal domains of ssSPTa and ssSPTb, which are highly conserved within each subfamily but are the most divergent regions between isoform subfamilies, are not required for activation of the heterodimer or for acyl-CoA selectivity suggests that the ssSPTs have additional roles that remain to be discovered

Objectives

Results

Conclusion