title: Membrane docking of an aggregation-prone protein improves its solubilization.
abstract: We used preS2-S'-beta-galactosidase, a three domain fusion protein that aggregates extensively at 43 degrees C in the cytoplasm of Escherichia coli to search for multicopy suppressors of protein aggregation and inclusion bodies formation, and took advantage of the known differential solubility of preS2-S'-beta-galactosidase at 37 and 43 degrees C to develop a selection procedure for the gene products that would prevent its aggregation in vivo at 43 degrees C. First, we demonstrate that the differential solubility of preS2-S'-beta-galactosidase results in a lactose-positive phenotype at 37 degrees C as opposed to a lactose-negative phenotype at 43 degrees C. We searched for multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C by selecting pink lactose-positive colonies on a background of white lactose-negative colonies after transformation of bacteria with an E. coli gene bank. We found only two multicopy suppressors of preS2-S'-beta-galactosidase aggregation at 43 degrees C, protein isoaspartate methyltransferase (PIMT) and the membrane components ChbBC of the N,N'-diacetylchitobiose phosphotransferase transporter. We have previously shown that PIMT overexpression reduces the level of isoaspartate in preS2-S'-beta-galactosidase, increases its thermal stability and consequently helps in its solubilization at 43 degrees C (Kern et al., J. Bacteriol. 187, 1377-1383). In the present work, we show that ChbBC overexpression targets a fraction of preS2-S'-beta-galactosidase to the membrane, and decreases its amount in inclusion bodies, which results in its decreased thermodenaturation and in a lactose-positive phenotype at 43 degrees C. Cross-linking experiments show that the inner membrane protein ChbC interacts with preS2-S'-beta-galactosidase. Our results suggest that membrane docking of aggregation-prone proteins might be a useful method for their solubilization.
authors.affiliation: Molecules de stress, Institut Jacques Monod, Université Paris 7, 2, place Jussieu, 75005 Paris, France.
authors.forename: Jihen
authors.initials: J
authors.lastname: Tagourti
authors.forename: Abderrahim
authors.initials: A
authors.lastname: Malki
authors.forename: Renée
authors.initials: R
authors.lastname: Kern
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: d'Alençon
authors.forename: Gilbert
authors.initials: G
authors.lastname: Richarme
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism*; D007785:Lactose / Q000378:metabolism; D008954:Models, Biological; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013696:Temperature; D001616:beta-Galactosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; D007785:Lactose