Localization of glycerophosphate acyltransferase in Escherichia coli

Abstract

sn-Glycero-3-phosphate acyltransferase (EC 2.3.1.15) the first enzyme involved in phospholipid biosynthesis, is known to be associated with the cytoplasmic membrane of Escherichiu cofi. The localization of this enzyme in the transverse plane of the membrane was investigated by proteolysis of intact and lysed spheroplasts and by inhibition of glycerol 3-phosphate transport into intact cells in the presence of azide. Glycerophosphate acyltransferase was found to be resistent to proteolysis by trypsin in intact spheroplasts, whereas its enzymatic activity could be destroyed completely by trypsin in lysed spheroplasts. These results are in line with a localization of the acyltransferase at the inner aspect of the cytoplasmic membrane. Sodium azide was shown to have no inhibitory effect on glycerophosphate acyltransferase activity. Lack of incorporation of glycero phosphate into the phospholipids of glycerol phosphate transport-negative cells and inhibition of this incorporation in wild-type and glycerol 3-phosphate transport-constitutive cells by azide support a cytoplasmic-oriented localization of the glycerophosphate acyltransferase.

Studies on reconstituted partially purified glycerophosphate acyltransferase from Escherichia coli

“…We have previously shown that in this membrane [7] as well as in reconstituted model membranes [5] all active sites of the glycerophosphate acyltransferase are present at one side of the membrane. The latency of the enzyme in E. coli spheroplasts [7] indicates absence of acylation when both substrates are present at the side of the membrane opposite to the active site. Here we investigate whether the glycerophosphate acyltransferase can function properly when each of the substrates is present at a different side of the membrane.…”