Membrane topology analysis of the Escherichia coli cytosine permease.
The Escherichia coli codBA operon encodes cytosine permease (CodB) and cytosine deaminase (CodA). CodB mediates uptake of exogenously supplied cytosine, and CodA catalyses the hydrolytic deamination of cytosine to uracil and ammonia. The hydropathic profile of CodB indicates that it is an integral cytoplasmic membrane protein possessing several transmembrane-spanning domains. The membrane topology of CodB was investigated by using gene fusions containing varying lengths of the amino-terminus of CodB fused to either alkaline phosphatase (AP) or beta-galactosidase (BG). The AP activities expressed by the CodB-AP fusions are consistent with a topological model in which the amino- and the carboxy-termini of CodB are located in the cytoplasm, and in which CodB possesses 12 membrane-spanning segments. The enzyme activities of most of the CodB-BG fusions support the model. However, the results obtained with some of the CodB-BG fusions illustrate the limitations of using BG as a reporter protein in studies of membrane protein topology.
Danielsen
S
S
Department of Biological Chemistry, University of Copenhagen, Denmark.
Boyd
D
D
Neuhard
J
J
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C075818:codB protein, E coli; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase