title: Extracellular labeling of nascent polypeptides traversing the membrane of Escherichia coli.
abstract: 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.
mesh_terms: D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004792:Enzyme Precursors / Q000096:biosynthesis; D004926:Escherichia coli / Q000378:metabolism*; D011132:Polyribosomes / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D013450:Sulfones
chemical_list: D001426:Bacterial Proteins; D004792:Enzyme Precursors; D013450:Sulfones; D012343:RNA, Transfer; D000469:Alkaline Phosphatase