The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the λP(L) and tac promoters. Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained. Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-Å diameter tubular structures associated with the cytoplasmic membrane. These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase. Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions. At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 Å from the cytoplasmic surface. Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 Å) and (1/39 Å) along the tubule axis and a prominent spot at (1/62 Å) near the equator. From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - Dec 1 1986|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology