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

AUTOR(ES)

Smith, W P

RESUMO

To provide direct evidence for the hypothesis that secreted proteins may traverse membranes as growing chains, we labeled spheroplasts of Escherichia coli 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 found to be 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 molecular weight, on peptide chain completion. Moreover, a significant fraction of the completed chains was identified serologically and by molecular weight as a major periplasmic protein, alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum); EC 3.1.3.1]. This work provides direct evidence that: (i) secreted proteins thread through the membrane as growing peptide chains; and (ii) membrane-associated polysomes in bacteria are functionally attached to membrane and not merely trapped on disruption of the cell.

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