Abstract
The first and committed step in synthesis of the ceramide moiety of sphingolipids is catalyzed by serine palmitoyltransferase (EC 2.3.1.50), which condenses palmitoyl-CoA and serine to form 3-ketosphinganine. This step is thought to be tightly regulated to control the synthesis of sphingolipids, but data supporting this hypothesis are lacking mainly because the enzyme has resisted purification and consequent characterization. Rather than attempting to purify the enzyme from normal cells, we have taken a different tack and opted to try and overproduce the enzyme to facilitate its purification. Here we demonstrate that overproduction in Saccharomyces cerevisiae requires expression of LCB1, a previously isolated yeast gene, and LCB2, the isolation and characterization of which we describe. Several lines of evidence argue that both genes encode subunits of the enzyme; however, biochemical evidence will be needed to substantiate this hypothesis. Although overproduction was modest, 2- to 4-fold, it should now be possible to devise improved overproduction vectors for yeast or other host organisms.
MeSH Terms
Acyltransferases/biosynthesis,genetics,metabolism
Amino Acid Sequence
Gene Expression
Genes, Fungal
Genotype
Macromolecular Substances
Molecular Sequence Data
Plasmids
Restriction Mapping
Saccharomyces cerevisiae/enzymology,genetics
Saccharomyces cerevisiae Proteins
Sequence Homology, Amino Acid
Serine C-Palmitoyltransferase
Sphingolipids/biosynthesis
Chemicals
Macromolecular Substances
Saccharomyces cerevisiae Proteins
Sphingolipids
Acyltransferases
LCB1 protein, S cerevisiae
LCB2 protein, S cerevisiae
Serine C-Palmitoyltransferase
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Nagiec M M
Department of Biochemistry, University of Kentucky, Lexington 40536-0084.
Baltisberger J A
Wells G B
Lester R L
Dickson R C
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