Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli.

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Abstract
To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, spheroplasts of E. coli were labeled with a reagent (acetyl[35S]methionyl methylphosphate sulfone) that reacts with amino groups but does not cross the membrane. After fractionation, about 6% of the label in the membrane-polysome fraction was attached to the polysomes. This attachment was via peptidyl-tRNA, as shown by several tests: release of most of the label from purified polysomes at low Mg2+; subsequent loss of about 25,000 daltons on cleavage by dilute alkali; release by puromycin; and release, accompanied by a marked increase in average MW, on peptide chain completion. A significant fraction of the completed chains was identified serologically and by MW as a major periplasmic protein, alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1]. Apparently secreted proteins thread through the membrane as growing peptide chains and membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.