Abstract
The orientation of the major coat (B) protein of the bacteriophage f1, an integral membrane protein in the cytoplasmic membrane of infected Escherichia coli, was examined. Pyridoxal 5'-phosphate and [3H]NaBH4 were used to label the cytoplasmic membrane proteins in spheroplasts and membrane vesicles of E. coli infected with bacteriophage f1. Under the conditions described, tritium incorporation was almost completely dependent on the presence of pyridoxal 5'-phosphate and little if any of the cytoplasmic proteins were labeled when the reaction was applied to intact spheroplasts. The major coat protein was isolated from the cytoplasmic membranes labeled in this manner and the chymotryptic peptides were analyzed for the presence of tritium in the pyridoxamine 5'-phosphate conjugate. When the proteins were labeled in the intact spheroplast, only the NH2-terminal chymotryptic peptide of the coat protein was labeled. If the proteins were labeled during osmotic lysis of the spheroplasts or in isolated vesicles, the chymotryptic peptide containing the COOH terminus of the coat protein as well as the NH2-terminal peptide was labeled. The NH2-terminal peptide was labeled to approximately the same extent as occurred in the intact spheroplast. These results are consistent with the hypothesis that the mature f1 coat protein asymmetrically spans the cytoplasmic membrane of the infected host with its NH2 terminus exposed on the outside and COOH terminus exposed on the cytoplasmic surface.
Highlights
Cytoplasmic membraneof infectedEscherichia CoZi,was Some of these studies have suggested that synthesis of the examined
Osmotic lysis of the spheroplastsor in isolatedvesicles, The observation that antibodies directed against the NHtthe chymotryptic peptide containing theCOOH termi- terminal portion of the coat protein bind to cells or spheronus of thecoatprotein as well as the Mia-terminal plasts of infected bacteria is supportive of this hypothesis [24]
There are two hydrophilic regions composed of the NHz-terminal 20 amino acids and the COOHterminal 11 amino acids which interact with the aqueous environment outside the membrane and are susceptible to cleavage by proteolytic enzymes (Fig. 1)
Summary
[3H]NaBH4(64 and 7.6 Ci/mmol), ~-['~C]lysine(342 mCi/mmol), ~-[4,5-~H]lysin(e78.1. 3 ml of 25 mM NazB407,pH 8.0, the unbroken cells were removed by centrifugation at lo00 X g for 20 min, and the membranes were. Ci/mmol), ~-['~C]proline(288 mCi/mmol), and Protosol were pur- collected by centrifugation as described above. The bottom 0.35 ml chased from New England Nuclear. Both the tritiated and nonradio- was collected from each sample and treatedwith 2 pl of 0.1 M NaBH4 active NaBH, were dissolved in 10 mM NaOH and used fresh or stored at -70 "C. The membranes in sample C were reacted with PLP and ['HINaBHr and those in sample D were reacted with buffer and [3H]NaBH4 exactly as delate was purchased from Fisher Scientific and recrystallized from scribed above for the intacstpheroplasts. Dialysis tubing was purchased from Spectrum Medical Industries, Inc., and Bio-Gel P-2 was from through a 23-gaugeneedle after the addition of each reagent to ensure the availability of the reactants toboth sides of the membrane
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