Cloning and characterization of LCB1, a Saccharomyces gene required for biosynthesis of the long-chain base component of sphingolipids.
The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.
Buede
R
R
Department of Biochemistry, University of Kentucky, Lexington 40536.
Rinker-Schaffer
C
C
Pinto
W J
WJ
Lester
R L
RL
Dickson
R C
RC
D000217:Acyltransferases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D051102:Serine C-Palmitoyltransferase; D013107:Sphingolipids / Q000096:biosynthesis*; D014158:Transcription, Genetic
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D013107:Sphingolipids; D000217:Acyltransferases; C495086:LCB1 protein, S cerevisiae; D051102:Serine C-Palmitoyltransferase