Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases.
We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.
Brown
A P
AP
Department of Biological Sciences, University of Durham, UK.
Coleman
J
J
Tommey
A M
AM
Watson
M D
MD
Slabas
A R
AR
D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D015723:Gene Library; D017343:Genes, Plant / Q000235:genetics; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D010743:Phospholipids / Q000032:analysis; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D003313:Zea mays / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D010743:Phospholipids; D000217:Acyltransferases; D051103:1-Acylglycerol-3-Phosphate O-Acyltransferase; C495091:plsC protein, E coli