Alain Rigal

Distinct regions of the colicin A translocation domain are involved in the interaction with TolA and TolB proteins upon import into Escherichia coli

Group A colicins need proteins of the Escherichia coli envelope Tol complex (TolA, TolB, TolQ and TolR) to reach their cellular target. The N‐terminal domain of colicins is involved in the import process. The N‐terminal domains of colicins A and E1 have been shown to interact with TolA, and the N‐terminal domain of colicin E3 has been shown to interact with TolB. We found that a pentapeptide conserved in the N‐terminal domain of all group A colicins, the ‘TolA box’, was important for colicin A import but was not involved in the colicin A–TolA interaction. It was, however, involved in the colicin A–TolB interaction. The interactions of colicin A N‐terminal domain deletion mutants with TolA and TolB were investigated. Random mutagenesis was performed on a construct allowing the colicin A N‐terminal domain to be exported in the bacteria periplasm. This enabled us to select mutant protein domains unable to compete with the wild‐type domain of the entire colicin A for import into the cells. Our results demonstrate that different regions of the colicin A N‐terminal domain interact with TolA and TolB. The colicin A N‐terminal domain was also shown to form a trimeric complex with TolA and TolB.

Structure of the Escherichia coli TolB protein determined by MAD methods at 1.95 Å resolution

BACKGROUND The periplasmic protein TolB from Escherichia coli is part of the Tol-PAL (peptidoglycan-associated lipoprotein) multiprotein complex used by group A colicins to penetrate and kill cells. TolB homologues are found in many gram-negative bacteria and the Tol-PAL system is thought to play a role in bacterial envelope integrity. TolB is required for lethal infection by Salmonella typhimurium in mice. RESULTS The crystal structure of the selenomethionine-substituted TolB protein from E. coli was solved using multiwavelength anomalous dispersion methods and refined to 1. 95 A. TolB has a two-domain structure. The N-terminal domain consists of two alpha helices, a five-stranded beta-sheet floor and a long loop at the back of this floor. The C-terminal domain is a six-bladed beta propeller. The small, possibly mobile, contact area (430 A(2)) between the two domains involves residues from the two helices and the first and sixth blades of the beta propeller. All available genomic sequences were used to identify new TolB homologues in gram-negative bacteria. The TolB structure was then interpreted using the observed conservation pattern. CONCLUSIONS The TolB beta-propeller C-terminal domain exhibits sequence similarities to numerous members of the prolyl oligopeptidase family and, to a lesser extent, to class B metallo-beta-lactamases. The alpha/beta N-terminal domain shares a structural similarity with the C-terminal domain of transfer RNA ligases. We suggest that the TolB protein might be part of a multiprotein complex involved in the recycling of peptidoglycan or in its covalent linking with lipoproteins.

The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step.

Colicins use two envelope multiprotein systems to reach their cellular target in susceptible cells of Escherichia coli: the Tol system for group A colicins and the TonB system for group B colicins. The N‐terminal domain of colicins is involved in the translocation step. To determine whether it interacts in vivo with proteins of the translocation system, constructs were designed to produce and export to the cell periplasm the N‐terminal domains of colicin E3 (group A) and colicin B (group B). Producing cells became specifically tolerant to entire extracellular colicins of the same group. The periplasmic N‐terminal domains therefore compete with entire colicins for proteins of the translocation system and thus interact in situ with these proteins on the inner side of the outer membrane. In vivo cross‐linking and co‐immunoprecipitation experiments in cells producing the colicin E3 N‐terminal domain demonstrated the existence of a 120 kDa complex containing the colicin domain and TolB. After in vitro cross‐linking experiments with these two purified proteins, a 120 kDa complex was also obtained. This suggests that the complex obtained in vivo contains exclusively TolB and the colicin E3 domain. The N‐terminal domain of a translocation‐defective colicin E3 mutant was found to no longer interact with TolB. Hence, this interaction must play an important role in colicin E3 translocation.

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes

SummaryIn vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.

Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm

Several proteins of the Tol/Pal system are required for group A colicin import into Escherichia coli. Colicin A interacts with TolA and TolB via distinct regions of its N‐terminal domain. Both interactions are required for colicin translocation. Using in vivo and in vitro approaches, we show in this study that colicin A also interacts with a third component of the Tol/Pal system required for colicin import, TolR. This interaction is specific to colicins dependent on TolR for their translocation, strongly suggesting a direct involvement of the interaction in the colicin translocation step. TolR is anchored to the inner membrane by a single transmembrane segment and protrudes into the periplasm. The interaction involves part of the periplasmic domain of TolR and a small region of the colicin A N‐terminal domain. This region and the other regions responsible for the interaction with TolA and TolB have been mapped precisely within the colicin A N‐terminal domain and appear to be arranged linearly in the colicin sequence. Multiple contacts with periplasmic‐exposed Tol proteins are therefore a general principle required for group A colicin translocation.

Aminopeptidase N- and human blood group A-antigenicity along the digestive tract and associated glands in the rabbit

SummaryThe cellular localization of an aminopeptidase N homologous to the brush-border intestinal enzyme and that of human blood group A-substances were investigated using the immunofluorescence technique on thin frozen sections (200 nm) of the digestive tract and associated glands of A+ and A− rabbits. Aminopeptidase N was found to be a common specific marker of both the apical region of plasma membrane of acinar cells in submaxillary and parotid glands and pancreas and the brush border of jejunum and colon absorbing cells. In hepatocytes, the enzyme was localized in the sinusoidal domains. Soluble A-substances were present in mucus secretory granules of intestinal goblet cells and those of stomach and gall bladder mucous cells. In contrast, the mucous acini of sublingual and submaxillary glands were devoid of A-antigenicity. The columnar cells of striated ducts of these glands exhibited A-antigenicity. Soluble A-substances were also found in zymogen granules of parotid and pancreas acinar cells and those of stomach chief cells. Moreover, in all cells secreting A-substances, and in the non-secreting absorbing intestinal cells, the glycoproteins of the plasma membrane bore A-determinants. Aminopeptidase N was one of the membrane-bound glycoproteins that bore A-determinants in cells that expressed A-antigenicity.

Subcellular fractionation and subcellular localization of aminopeptidase N in the rabbit enterocytes

SummaryA fast and easy procedure is proposed for preparing concomitantly from the same sample of intestinal mucosa of A+ rabbits, four fractions high enriched in the brush-border and basolateral plasma membrane domains, rough endoplasmic reticulum, and smooth endoplasmic reticulum plus Golgi apparatus membranes, respectively. This is the first time the technique of flow fluorometry has been applied to characterize the brush-border and basolateral membrane fractions using polyclonal or monoclonal antibodies against antigens common to or specific for these two plasma membrane domains. This technique definitely proves the presence of aminopeptidase in at least 60% of the basolateral membrane vesicles, where its level is about 4.5% of that in the brush-border membrane vesicles. The endoglycosidase H-sensitive intermediate of glycosylation of aminopeptidase N in the steady state is accumulated in both the rough and smooth endoplasmic reticulum membranes. Although the rough membrane is more extensive it contains only about 40% of this transient form.

Cellular and subcellular localization of annexin IV in rabbit intestinal epithelium, pancreas and liver

Summry— The results of immunoblot analysis performed with a specific monoclonal antibody showed that the intestinal mucosa, pancreas and liver are privileged tissues for the expression of annexin IV. Immunofluorescence labelling of thin frozen sections of these tissues showed a strong concentration of annexin IV along the basolateral domain of the plasma membrane of intestinal absorbing cells, hepatocytes and pancreatic acinar cells, whereas in intestinal mucous secreting cells and centro acinar pancreatic cells, annexin IV was found to be present throughout the cytoplasm.

Biosynthesis and intracellular pool of aminopeptidase N in rabbit enterocytes

SummaryA papain treatment at 15°C and pH 7.3 of a microsomal fraction from rabbit enterocytes quantitatively releases the aminopeptidase N integrated in the plasma membranes without solubilizing the enzyme integrated in the intracellular membranes. Working on A+ rabbits, characterized by the presence on the brush-border hydrolases of glycans corresponding to the human blood group A-determinant structure, it was possible to separate the intracellular aminopeptidase into two major molecular forms with or without these determinants. The molecular form devoid of human blood group A antigenicity corresponds to the only stable intermediate of glycosylation, bearing N-linked high mannose oligosaccharides. This endoglycosidase H-sensitive form is fully active and represents in the steady state about 1% of the total cellular aminopeptidase. It contains a cytoplasmic sequence of about 3000 daltons that has not yet been detected in the mature form. The A antigenicity is acquired simultaneously with processing of high mannose glycans to complex glycans. Pulse chase labeling of jejunum loops with [35S]-methionine showed that the complete processing of the transient form synthesized during 10 min takes 1 hr. During the last 30 min of processing, all the newly transformed molecules are transported to the plasma membrane.

High-Order ADI Schemes for Convection-Diffusion Equations with Mixed Derivative Terms

We consider new high-order Alternating Direction Implicit (ADI) schemes for the numerical solution of initial-boundary value problems for convection-diffusion equations with cross derivative terms. Our approach is based on the unconditionally stable ADI scheme proposed by Hundsdorfer. Different numerical discretizations which lead to schemes which are fourth-order accurate in space and second-order accurate in time are discussed.