Properties and organization of the proteins in the outer membrane of Escherichia coli

Two major proteins of the outer membrane of Escherichia coli, the matrix protein, A (M.W. 36,500) and the heat-modifiable protein, B were purified and partially characterized. Both have a low content of cysteine, . an excess of acidic amino acids over basic and a moderate content of hydrophobic amino acids. Protein B (M.W. 28,500) was converted to form B* (M.W. 33j^00) upon heating in the presence of sodium dodecyl sulfate at temperatures higher than 50°C. Physical studies showed that protein B unfolds upon heating without a large increase in binding of sodium dodecyl sulfate. It is proposed that protein B as extracted from the membrane contains some native structure which is lost upon heating. The level of protein A1, a major outer membrane protein in glucose-grown cells, was decreased in cells grown on other carbon sources with a concomitant increase in the amount of protein A2. Both proteins were tightly associated with the peptidoglycan and had similar amino acid composition, suggesting that they play the same role in the outer membrane. The organization of proteins in the outer membrane of E. coli was studied by proteolytic digestion, covalent labelling and crosslinking. The proteins of the outer membrane were inaccessible to pronase in intact cells and the cells altered in their lipopolysaccharide component. The protein components of isolated outer membrane preparations varied in their rates of digestion and labelling with fluoresca- ' mine, suggesting that they are asymmetrically arranged in the membrane. The proteins most rapidly degraded (proteins B,C-,D1 and E) were judged to be exposed at the surface of the membrane, while those resistant to digestion (proteins A1,A2 and D2) must be protected by their arrangement in the membrane. Digestion of outer membrane preparations with pronase left a fragment derived from protein B (protein Bp) embedded in the membrane. This fragment was not enriched in hydrophobic amino acids relative to protein B. Protein B could be reassociated with itself j without phospholipid or lipopolysacchari.de such that pronase digestion of the reassociated material gave protein Bp. These results suggest that protein B may not be held in the membrane primarily by hydrophobic interactions. The resistance of proteins A1 and A2 to protease digestion is likely due to protein-protein interactions since oligomers of protein A could be isolated. Treatment of protein A1- or A2-peptidogly-can complexes with dithiobis (succinimidyl propionate) or glutaraldehyde produced dimer, trimer and higher oligomers of protein A. No crosslinking of protein A to the peptidoglycan was detected. The proteins of the isolated outer membrane varied in their ease of crosslinking. Protein B, but not the pronase-resistant fragment, protein Bp, was readily crosslinked to give high molecular weight oligomers, while protein A formed dimers and trimers under the same conditions. No crosslinking of protein A to B was detected. Crosslinking of cell wall preparations showed that protein B and the free form of the lipoprotein, F, could be linked to the peptidoglycan. A dimer of protein F, and protein F linked to protein B, were detected. These results suggest that specific protein-protein interactions occur in the outer membrane. A model for the arrangement of the proteins in the outer membrane of E. coli, summarizing the results of proteolytic digestion, covalent labelling and crosslinking, is presented.Medicine, Faculty ofBiochemistry and Molecular Biology, Department ofGraduat

Palmitoylation is not required for trafficking of human anion exchanger 1 to the cell surface.

AE1 (anion exchanger 1) is a glycoprotein found in the plasma membrane of erythrocytes, where it mediates the electroneutral exchange of chloride and bicarbonate, a process important in CO2 removal from tissues. It had been previously shown that human AE1 purified from erythrocytes is covalently modified at Cys-843 in the membrane domain with palmitic acid. In this study, the role of Cys-843 in human AE1 trafficking was investigated by expressing various AE1 and Cys-843Ala (C843A) mutant constructs in transiently transfected HEK-293 cells. The AE1 C843A mutant was expressed to a similar level to AE1. The rate of N-glycan conversion from high-mannose into complex form in a glycosylation mutant (N555) of AE1 C843A, and thus the rate of trafficking from the endoplasmic reticulum to the Golgi, were comparable with that of AE1 (N555). Like AE1, AE1 C843A could be biotinylated at the cell surface, indicating that a cysteine residue at position 843 is not required for cell-surface expression of the protein. The turnover rate of AE1 C843A was not significantly different from AE1. While other proteins could be palmitoylated, labelling of transiently transfected HEK-293 cells or COS7 cells with [3H]palmitic acid failed to produce any detectable AE1 palmitoylation. These results suggest that AE1 is not palmitoylated in HEK-293 or COS7 cells and can traffic to the plasma membrane

Impaired trafficking of human kidney anion exchanger (kAE1) caused by hetero-oligomer formation with a truncated mutant associated with distal renal tubular acidosis.

Autosomal dominant distal renal tubular acidosis (dRTA) has been associated with several mutations in the anion exchanger AE1 gene. The effect of an 11-amino-acid C-terminal dRTA truncation mutation (901 stop) on the expression of kidney AE1 (kAE1) and erythroid AE1 was examined in transiently transfected HEK-293 cells. Unlike the wild-type proteins, kAE1 901 stop and AE1 901 stop mutants exhibited impaired trafficking from the endoplasmic reticulum to the plasma membrane as determined by immunolocalization, cell-surface biotinylation, oligosaccharide processing and pulse-chase experiments. The 901 stop mutants were able to bind to an inhibitor affinity resin, suggesting that these mutant membrane proteins were not grossly misfolded. Co-expression of wild-type and mutant kAE1 or AE1 resulted in intracellular retention of the wild-type proteins in a pre-medial Golgi compartment. This dominant negative effect was due to hetero-oligomer formation of the mutant and wild-type proteins. Intracellular retention of kAE1 in the alpha-intercalated cells of the kidney would account for the impaired acid secretion into the urine characteristic of dRTA

Band 3 structure.

<p>A. Structure of the dimeric Band 3 membrane domain (mdAE1) that was used in our simulations. The mdAE1 core domain is shown in grey and the gate domain in orange. Note that some unstructured regions that are missing from the crystal structure (PDB: 4YZF) have been modelled. B. Snapshot from the end of one of the atomistic simulations in which mdAE1 is embedded in a complex asymmetric bilayer (Band3_AT-1). The different lipid types are shown in different colors and the water is shown in ice-blue.</p