The Topology of the Lcb1p Subunit of Yeast Serine Palmitoyltransferase

Abstract

The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of ϳ60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the ␣-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.

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

“…We have shown that transfection of mammalian cells with the human LCB1 ortholog (SPTLC1) and the LCB2-like isoform, SPTLC2, produces only a modest increase in SPT activity, despite high levels of protein expression (11). The same is true for cotransfection of SPTLC1 and the other LCB2-like isoform, SPTLC3 (9).…”

“…3B). These results suggest that endogenous levels of ssSPTa limit SPT activity, thus providing an explanation for the lack of a simple correlation between levels of hLCB1 and hLCB2a/hLCB2b expression and SPT activity (11).…”

“…Although coexpression of hLCB1 with either hLCB2 subunit in mammalian cells increases SPT activity, the increased activity does not correlate well with the levels of subunit expression (11). In addition, we have recently coexpressed the LCB1 and LCB2 subunits from a variety of higher eukaryotes in lcb1⌬ lcb2⌬ yeast and found none conferred activity comparable to that measured in the organisms of origin (8,19).…”

“…opportunity to examine each potential multimeric SPT complex in the absence of any endogenous SPT subunits or activity. All 5 human SPT subunits could be stably expressed in yeast, although as has been reported (11,16), hLCB2a expression required coexpression of hLCB1 (Fig. 5A).…”

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activities

“…The topology of ScORM2 protein was mapped based on the previously described methodology (Han et al, 2004). In brief, NheI restriction sites were created before codons for amino acids 2, 100, 135, and 169 and an XbaI site before the stop codon (217) by use of the QuikChange mutagenesis kit (Agilent Technology).…”