f1 Coat protein synthesis and altered phospholipid metabolism in f1 infected Escherichia coli

The coat protein of a filamentous phage (M13) enters the cytoplasmic membrane from two directions: from the outside upon infection and from the cell interior late in the viral life cycle prior to phage assembly and extrusion. Binding of 125I-labeled anti-coat protein antibody to spheroplasts or to inverted vesicles was used to assay the orientation of coat protein in the membrane. Both parental and newly synthesized coat protein were found to be exposed on the outer surface of the cytoplasmic membrane. Coat protein in intact infected cells is also accessible to external antibody. Thus two different processes of assembling a protein into membrane, each starting from a different membrane surface, appear to produce similar surface orientations.Several striking features of membrane structure and function raise questions as to how this organelle is assembled. Integral membrane proteins are hydrophobic and tend to aggregate in the absence of detergent (1, 2), in contrast to the hydrophilic character of the known machinery of protein synthesis. Different proteins, and perhaps lipids, are oriented asymmetrically towards the inner or outer surface of the bilayer (3-6), despite the apparent localization of protein and lipid synthetic enzymes to inside the cell and despite the very low rates at which lipids and proteins "flip" through the hydrocarbon membrane core (7).The M13 coliphage offers several apparent advantages for the study of these assembly problems. The filamentous M13 virion consists of a single-stranded circular DNA of 2 X 106 daltons (8) encapsulated in a helical array of coat proteins (9). The coat protein is a 5260 dalton peptide of known sequence with a central hydrophobic region (10). This small peptide is 98% of the virion protein (8). Each virion also has two to four copies of a 70,000 dalton protein, important in several stages of viral growth (11). When Mi3 infects a male cell, all of the parental coat protein enters the cytoplasmic membrane as the viral DNA is replicated (12)(13)(14). This parental coat protein is later incorporated dispersively into progeny virus (15). Late in the infectious process, new coat protein is made and inserted into the cytoplasmic membrane at a prodigious rate, reaching 26% of the membrane protein synthesis (13). Viral DNA, extruding through the membrane, is encapsulated by these coat protein molecules. Approximately 1000 virus particles, each with 1800 coat protein molecules, are made during each cell generation (8). The extraordinary rate of synthesis of this small, defined peptide and the existence of two means of inserting it into membrane, one during infection and one during phage production, recommend it as a system for the study of membrane protein synthesis and assembly.This communication reports the development of rapid immunological assays for the protein, both in the membranebound state and as solubilized by detergents. Using these as- (16) at 370 and infected with phage at a multiplicity of 2. After 9 hr of growth, cells were removed by continuous fl...

supporting

confidence: 78%

Asymmetric orientation of a phage coat protein in cytoplasmic membrane of Escherichia coli.

Wickner¹

1975

“…Although produced in modest amounts, these proteins, which associate with the inner membrane, are lethal to the host under conditions where wild-type pIV produced by the parental plasmid is not (data not shown). Membrane perturbation, itself, does not prevent induction of Psp synthesis, since gene I and gene IV amber mutant infections exhibit similar membrane disturbances (33,34), and Psp induction does occur in the former.…”

Section: Methodsmentioning

confidence: 99%

“…Cells infected by gene I or gene IV amber mutants accumulate virion structural proteins, suffer membrane hyperplasia, and eventually die (33,34 Although Psp is induced by heat shock, it is not a conventional heat shock protein. Its full induction requires more extreme treatments than induction of the classical heat shock proteins and is independent of the heat shock oa factor (&32).…”

mentioning

confidence: 99%

Phage shock protein, a stress protein of Escherichia coli.

Brissette

Russel

Weiner

et al. 1990

235

260

Filamentous phage infection induces the synthesis of large amounts of an Escherichia coli protein, phage shock protein (Psp), the product of a previously undescribed gene. This induction is due to the phage gene IV protein, pIV, an integral membrane protein. The uninduced level of Psp is undetectable, but when induced by prolonged synthesis of pIV, it can become one of the most abundant proteins in the cell. Psp is also synthesized transiently in response to several stresses (heat, ethanol, and osmotic shock). High-level synthesis occurs only after extreme treatment. Unlike the members of the heat shock regulon, Psp induction does not require the heat shock sigma factor, sigma 32; some stimuli that elicit sigma 32-dependent heat shock proteins do not induce Psp synthesis. The level of Psp induction after extreme stress is even higher in sigma 32 mutant cells, which are unable to mount a normal heat shock response, suggesting that these parallel stress responses are interrelated.

“…The identification of most phage products has been hampered by this continuation of host growth. However, products of the phage genes V and VIII (coat protein) are synthesized in large quantities and can be easily identified (3,4). The two proteins are also the major products of coupled transcription-translation directed in vitro by replicative form DNA (5,6).…”

mentioning

confidence: 99%

Bacteriophage f1 infection of Escherichia coli: identification and possible processing of f1-specific mRNAs in vivo.

Cashman¹,

Webster²

1979

ABSTRACT[3HJUracil-pulse-labeled RNA from Eschenichia coli infected with fl bacteriophage was fractionated on polyacrylamide gels containing urea. Eight phage-specific RNA species were present with approximate lengths ranging from 2100 to 400 nucleotides. The amount of the seven largest species was increased when the infected bacteria were incubated at 420C. When the RNA was isolated and used as message in an in vitro protein-synthesizing system, most of the RNA species appeared to direct the synthesis of the phage gene VIII protein.The six largest species also directed the synthesis of the phage gene V protein. Some of the labeled smaller RNA species increased in amount after addition of rifampicin, suggesting that they may have resulted from cleavage of larger RNA species. These particular smaller RNA species also were present in infected bacteria containing a mutant RNase III. The data are discussed in terms of the regulation of synthesis of the phagespecific proteins.The closely related filamentous bacteriophage f1, fd, and M13 contain a single-stranded DNA that codes for approximately eight genes (1, 2). Escherichia coli infected with these phage continue to grow and divide, while newly synthesized phage are extruded through the membrane. The identification of most phage products has been hampered by this continuation of host growth. However, products of the phage genes V and VIII (coat protein) are synthesized in large quantities and can be easily identified (3, 4). The two proteins are also the major products of coupled transcription-translation directed in vitro by replicative form DNA (5, 6). These findings suggest that there is some regulatory mechanism operating both in vivo and in vitro that allows this differential expression of phage products.Such a regulatory process may be operating partially at the level of transcription. Studies in vitro have shown that a small number of discrete transcripts are synthesized; initiation is from various promoters, while termination appears to occur at a unique site (7-11). Due to their location in the genome (12), the gene V and VIII regions are immediately proximal to the termination site; thus, most mRNAs in vitro would contain the coding information for these genes.In order to examine the basis for the high level of expression of the gene V and VIII proteins in vivo, it is necessary to characterize phage-specific mRNA found in infected bacteria. We report here the identification of a number of phage-specific mRNAs and show that several of them can direct the synthesis of the gene V and VIII proteins. Some of the species appear to correspond to those found in vitro, while others may have arisen as a result of processing from larger RNA molecules. These results are discussed in terms of the regulation of synthesis of the phage-specific proteins.MATERIALS AND METHODS E. coli K38, a nonsuppressing strain, and K37, a serine-inserting suppressing strain, were used (13). BL214, an RNase III-strain (14), was obtained from W. Studier. Bacteriophage f1, fi amber mutants...