Organization and stability of a polytopic membrane protein: deletion analysis of the lactose permease of Escherichia coli.
The overall topology of polytopic membrane proteins is thought to result from either the oriented insertion of the N-terminal alpha-helical domain followed by passive insertion of subsequent helices or from the function of independent topogenic determinants dispersed throughout the molecules. By using the lactose permease of Escherichia coli, a well-characterized membrane protein with 12 transmembrane domains and the N and C termini on the cytoplasmic surface of the membrane, we have studied the insertion and stability of in-frame deletion mutants. So long as the first N-terminal and the last four C-terminal putative alpha-helical domains are retained, stable polypeptides are inserted into the membrane, even when an odd number of helical domains is deleted. Moreover, even when an odd number of helices is deleted, the C terminus remains on the cytoplasmic surface of the membrane, as judged by lacY-phoA fusion analysis. In addition, permease molecules devoid of even or odd numbers of putative transmembrane helices retain a specific pathway for downhill lactose translocation. The findings imply that relatively short C-terminal domains of the permease contain topological information sufficient for insertion in the native orientation regardless of the orientation of the N terminus.
Bibi
E
E
Howard Hughes Medical Institute, Department of Physiology University of California, Los Angeles 90024-1574.
Verner
G
G
Chang
C Y
CY
Kaback
H R
HR
D000469:Alkaline Phosphatase / Q000378:metabolism; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000201:enzymology; D002872:Chromosome Deletion; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008961:Models, Structural; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016296:Mutagenesis; D011487:Protein Conformation; D015183:Restriction Mapping; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; C021499:lactose permease; D000469:Alkaline Phosphatase; D007785:Lactose