The role of L1 loop in the mechanism of rhomboid intramembrane protease GlpG.

Intramembrane proteases are important enzymes in biology. The recently solved crystal structures of rhomboid protease GlpG have provided useful insights into the mechanism of these membrane proteins. Besides revealing an internal water-filled cavity that harbored the Ser-His catalytic dyad, the crystal structure identified a novel structural domain (L1 loop) that lies on the side of the transmembrane helices.

Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease.

Rhomboids are a family of serine proteases belonging to intramembrane cleaving proteases, which are supposed to catalyse proteolysis of a substrate protein within the membrane. It remains unclear whether substrates of the rhomboid proteases have a common sequence feature that allows specific cleavage by rhomboids. We showed previously that GlpG, the Escherichia coli rhomboid, can cleave a type I model membrane protein Bla-LY2-MBP having the second transmembrane region of lactose permease (LY2) at the extramembrane region in vivo and in vitro, and that determinants for proteolysis reside within the LY2 sequence.

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.

Environment of the active site region of RseP, an Escherichia coli regulated intramembrane proteolysis protease, assessed by site-directed cysteine alkylation.

Regulated intramembrane proteolysis (RIP) plays crucial roles in both prokaryotic and eukaryotic organisms. Proteases for RIP cleave transmembrane regions of substrate membrane proteins. However, the molecular mechanisms for the proteolysis of membrane-embedded transmembrane sequences are largely unknown.

Proteolytic action of GlpG, a rhomboid protease in the Escherichia coli cytoplasmic membrane.

We characterized Escherichia coli GlpG as a membrane-embedded protease and a possible player in the regulated intramembrane proteolysis in this organism. From the sequence features, it belongs to the widely conserved rhomboid family of membrane proteases. We verified the expected topology of GlpG, and it traverses the membrane six times.