Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.

https://science.sciencemag.org/content/sci/233/4771/1403.full.pdf

A genetic approach to analyzing membrane protein topology

Previous studies support a model in which the physiological O2 gradient is transduced by haemoglobin into the coordinate release from red blood cells of O2 and nitric oxide (NO)-derived vasoactivity to optimize oxygen delivery in the arterial periphery1,2. But whereas both O2 and NO diffuse into red blood cells, only O2 can diffuse out3,4,5. Thus, for the dilation of blood vessels by red blood cells, there must be a mechanism to export NO-related vasoactivity, and current models of NO-mediated intercellular communication should be revised. Here we show that in human erythrocytes haemoglobin-derived S-nitrosothiol (SNO), generated from imported NO, is associated predominantly with the red blood cell membrane, and principally with cysteine residues in the haemoglobin-binding cytoplasmic domain of the anion exchanger AE1. Interaction with AE1 promotes the deoxygenated structure in SNO–haemoglobin, which subserves NO group transfer to the membrane. Furthermore, we show that vasodilatory activity is released from this membrane precinct by deoxygenation. Thus, the oxygen-regulated cellular mechanism that couples the synthesis and export of haemoglobin-derived NO bioactivity operates, at least in part, through formation of AE1–SNO at the membrane–cytosol interface.

Export by red blood cells of nitric oxide bioactivity.

The amino-acid sequence of murine band 3, deduced from the nucleotide sequence of a complementary DNA clone, confirms that this integral membrane glycoprotein is composed of two major structural domains which correlate with its dual functions as the anchor for the erythrocyte cytoskeleton and as a plasma membrane anion antiporter. This latter activity resides within a highly hydrophobic domain that crosses the plasma membrane at least 12 times.

Primary structure and transmembrane orientation of the murine anion exchange protein

Structure and function of the cytoplasmic domain of band 3: center of erythrocyte membrane-peripheral protein interactions.

To characterize the location of glycolytic enzymes (GEs) in intact human erythrocytes, freshly drawn blood was fixed and stained with Abs to GAPDH, aldolase, phosphofructokinase (PFK), pyruvate kinase (PK), lactate dehydrogenase (LDH), carbonic anhydrase II, Hb, and band 3 (AE1). Confocal microscopy revealed that in cells where band 3 displays its expected membrane staining and Hb is evenly distributed across the cytoplasm, GEs are largely limited to the membrane. Biochemical studies confirmed that the membrane binding sites for GAPDH, aldolase, and PFK reside on band 3, but related analyses demonstrate that sites for PK and LDH do not. Four lines of evidence demonstrate that the GEs are at least partially assembled into multimeric complexes near the NH2 terminus of band 3. First, a mAb to residues 1–12 of band 3 displaces all of the above GEs from the membrane, including LDH and PK, which do not bind band 3. Second, tyrosine phosphorylation of the NH2 terminus of band 3 (Y8 and Y21) reversibly releases all of the GEs from the membrane, including LDH and PK. Third, deoxygenation of RBCs dislodges all GEs from the membrane, consistent with the established ability of deoxyHb but not oxyHb to bind the NH2 terminus of band 3. Fourth, a large increase in the accessibility of enzyme epitopes is observed upon dissociation of GEs from the membrane. We conclude, therefore, that GEs are organized into complexes on the membrane whose assembly is regulated by oxygenation and phosphorylation.

https://www.pnas.org/content/pnas/102/7/2402.full.pdf

E. T. Kaiser

Papers

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane.