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

Abstract

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...

NMR Studies of the Major Coat Protein of Bacteriophage M13

“…It has intriguing properties in that it can exist as a cytoplasmic protein (procoat protein), as a structural element of the mature phage, and as an integral membrane protein at different stages of its lifecycle (Wickner, 1975;Ziinmerman et al, 1982;Rasched and Oberer, 1986). It is used as a model system for studying membrane assembly (Wickner, 1988).…”

Identifying Diagnostic Peptides for Lyme Disease through Epitope Discovery

“…Phage clones were propagated in 200-ml cultures, and virions were prepared at high purity as described previously (79). Wells of ELISA dishes were coated for at least 4 h at 4°C with 2 ϫ 10 10 virions in 50 l of TBS; washed with TBS-Tween on a plate washer; reacted for at least 2 h at 4°C with 50 l of individual serum samples (treated as described under "S/D treatment" above, but otherwise unprocessed) diluted 1:1,600 in TTDBA; washed; reacted for 1.5 h at room temperature with 50 l of AP-conjugated goat antibodies to human immunoglobulins (AP-anti-hIg, AP-anti-hIgG, or AP-antihIgM) diluted 1:2,500 in TTDBA; washed again; and developed with NPP substrate as described under "Phage capture ELISA" above.…”

Homogenous M13 Bacteriophage Quantification Assay Using Switchable Lanthanide Fluorescence Probes

“…Although titering by absorbance is a rapid technique compared with plating, very pure phage stocks are needed since protein impurities interfere with the measurement. For minimal impurities, phage should be purified using CsCl density-gradient ultracentrifugation, which requires centrifugation times up to 36 h (7). …”