The intramembrane active site of GlpG, an E. coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates.
Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.
Maegawa
Saki
S
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Koide
Kayo
K
Ito
Koreaki
K
Akiyama
Yoshinori
Y
D000477:Alkylating Agents / Q000494:pharmacology; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D029721:Drosophila Proteins / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D019897:Periplasm / Q000187:drug effects; D011092:Polyethylene Glycols / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism; D027981:Symporters / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000378:metabolism; D014867:Water / Q000378:metabolism*; D001618:beta-Lactamases / Q000378:metabolism
D000477:Alkylating Agents; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; C050221:LacY protein, E coli; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010455:Peptides; D013438:Sulfhydryl Compounds; D027981:Symporters; D016211:Transforming Growth Factor alpha; C083730:grk protein, Drosophila; D014867:Water; D011092:Polyethylene Glycols; D010450:Endopeptidases; D001618:beta-Lactamases; D005033:Ethylmaleimide