title: Design of a membrane protein for site-specific proteolysis: properties of engineered factor Xa protease sites in the lactose permease of Escherichia coli.
abstract: Lactose permease is a polytopic membrane transport protein with 12 hydrophobic transmembrane domains connected by hydrophilic loops on the cytoplasmic and periplasmic sides of the membrane. By the use of an active permease mutant devoid of Cys residues (C-less permease), single recognition sites (Ile-Glu-Gly-Arg) for the protease factor Xa (fXa) were engineered into hydrophilic loops 7, 8, and 10 in the C-terminal half of the protein. Mutants carrying single sites inserted at position 255, 259 (loop 7), 283, 286 (loop 8), or 341 (loop 10) exhibit significant lactose accumulation (30-70% of C-less permease) and normal levels of expression in the membrane. However, despite solubilization in dodecyl beta-D-maltoside, none of the mutant permeases is proteolyzed by fXa to a significant extent. Insertion of two recognition sites in tandem at position 255 results in partial cleavage, and remarkably, introduction of three sites in tandem leads to complete proteolysis by fXa. Importantly, mutants with two or three fXa sites at position 255 accumulate lactose to high levels (70% of C-less) and are present in the membrane in amounts comparable to that of C-less permease. The results indicate that hydrophilic loops 7, 8, and 10 are buried in the tertiary structure of the permease where they are inaccessible to protease. Insertion of tandem sites probably facilitates proteolysis by causing loops to become more accessible to the aqueous phase and by increasing the local concentration of protease recognition sites. The approach should be applicable to other polytopic membrane proteins.
authors: Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1570.
authors: M
authors: M
authors: Sahin-Tóth
authors: R L
authors: RL
authors: Dunten
authors: H R
authors: HR
authors: Kaback
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001693:Biological Transport, Active; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D027981:Symporters
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011994:Recombinant Proteins; D027981:Symporters; C021499:lactose permease; D015951:Factor Xa; D007785:Lactose