Membrane topography of ColE1 gene products: the immunity protein

Abstract

The topography of the colicin El immunity (Imm) protein was determined from the positions of TnphoA and complementary lacZ fusions relative to the three long hydrophobic segments of the protein and site-directed substitution of charged for nonpolar residues in the proposed membrane-spanning segments. Inactivation of the Imm protein function required substitution and insertion of two such charges. It was concluded that the 113-residue colicin El Imm protein folds in the membrane as three trans-membrane a-helices, with the NH2 and COOH termini on the cytoplasmic and periplasmic sides of the membrane, respectively. The approximate spans of the three helices are Asn-9 to . An extrinsic highly charged segment, Lys-66 to Lys-74, containing seven charges in nine residues, extends into the cytoplasmic domain. The specificity of the colicin El Imm protein for interaction with the translocation apparatus and the colicin El ion channel is proposed to reside in its peripheral segments exposed on the surface of the inner membrane. These regions include the highly charged segment Lys-66 to Lys-83 (loop 2) and the short (approximately eight-residue) NH2 terminus on the cytoplasmic side, and Glu-29 to Val-44 (loop 1) and the COOH-terminal segment Gly-105 to Asn-113 on the periplasmic side.Colicin El exerts its bactericidal effect by forming a highly conductive ion channel in the cytoplasmic membrane that depolarizes and deenergizes the cell (9). The channel domain, localized in the COOH-terminal third of the 522-residue colicin molecule, is susceptible to the protective effect of the 113-residue colicin El immunity (Imm) protein, a cytoplasmic membrane protein (3).Six different channel-forming colicins and their Imm proteins have been identified (26,28,(35)(36)(37) lacZ fusion takes advantage of P-galactosidase which, unlike AP, is active in the cytoplasm (29). In-frame lacZ fusions with a periplasmic domain of an integral membrane protein cause the lacZ gene product to be retained in the membrane, resulting in low 3-galactosidase activity (14). The dependence of lacZ fusion activity on the trans-membrane orientation of the fusion site is thus complementary to that of phoA fusions (29), so that this method can often, but not always (19), provide an additional determinant of the topography of integral membrane proteins. MATERIALS AND METHODSConstruction of plasmid pSL630. Plasmid pDMS630 (40), a ColEl derivative with an ampicillin resistance gene (constructed by insertion of Tn3 into ColEl) was digested with EcoRI and SmaI, each of which cuts at only one site. Two linear DNA fragments were generated, a 1.3-kb fragment that spans most of cea and a 10.5-kb fragment containing imm. These were blunt-ended by digestion with mung bean nuclease, self-ligated for 3 h at 16°C, and transformed into JM83. Colonies that grew on ampicillin were chosen randomly, plasmid DNA was isolated by a modified alkaline lysis method (34a), and a plasmid produced from self-ligation of the 10.5-kb fragment without the 1.3-kb fragment was isolated by d...

Membrane Topology and Insertion of Membrane Proteins: Search for Topogenic Signals

“…Positive LacZ fusions represent fusions to cytoplasmic domains, while positive PhoA fusions represent fusions to periplasmic domains. Genetic tricks have been developed which make it possible to exchange the LacZ reporter molecules in positive fusions for the PhoA molecule and vice versa (117,200), resulting in fusions with complementary enzymatic properties (76,170). The use of PhoA as a reporter molecule in these studies is advantageous in principle because a positive result (i.e., a highactivity fusion) requires the active export of the mature reporter enzyme moiety into the periplasm.…”

“…Various topology models have been tested using a combination of PhoA and LacZ fusions (45,57,76,91,92,106,170,202,206). Random fusions to both reporter molecules are often constructed, and this is followed by screen- ing for positive clones on indicator plates.…”

“…This systematic behavior can be used as an additional method to discriminate between fusions with a periplasmic or cytoplasmic PhoA moiety, but it is important that it is not used to normalize the activities. To demonstrate the synthesis of the fusion proteins, pulse and/or pulse-chase experiments are generally used (26,45,151,152,168,170,206,208).…”

Channel Domain of Colicin A Modifies the Dimeric Organization of Its Immunity Protein

“…The topological model of Cai, constructed from biochemical and genetic analyses (10,11), indicates the presence of four transmembrane segments. However, little was known about either the three-dimensional arrangement of the TMSs within the inner membrane or the oligomeric state of Cai.…”

“…The colicin A immunity protein (Cai) 2 has four transmembrane segments (TMSs), and its N and C termini are in the cytoplasm (Fig. 1), whereas the immunity protein to colicin E1 (Cei) crosses the cytoplasmic membrane three times, with the N terminus located in the cytoplasm and the C terminus in the periplasm (10,11).…”