Membrane reconstitution in chl-r mutants of Escherichia coli K 12. IX. Part played by phospholipids in the complementation process

Abstract

The supernatant extracts of the chl A and chl B mutants of Escherichia coli K 12, the phospholipids of which are labeled by growth in 32 P or [2- 3H]glycerol media, contain 20 times more radioactivity than the supernatant extract of the wild-type strain grown under the same conditions. We have observed that, after complementation, 80% of the radioactivity previously contained by Extracts A and B is incorporated into reconstituted particles. The chromatography of 3H-labeled Extract B on DEAE-cellulose and followed by gel filtration of radioactive fractions on Sephadex G-200 has shown that the phospholipids of Extract B are only bound to soluble proteins and not to fragments of membranes; it can be assumed that they have been solubilized in the form of a lipid-protein complex by cell breakage. When Extracts A and B are treated by phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) before being mixed together, an inhibition of the reconstitution of nitrate reductase activity which is proportional to the phospholipase C concentration and the length of treatment is observed. The analysis of lipids and phospholipids of particles (Peak I, Peak II and Peak III) formed during complementation and reconstituted nitrate reductase shows that their phospholipid contents (phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and phosphatidylserine) and especially that of Peak II (d equals 1.18) are closely related to that of native particles from the wild-type strain. These results allow one to propose a hypothesis explaining the mechanism involved in complementation.