Membrane-dependent cleavage of the human placental lactogen precursor to its native form in ascites cell-free extracts

Messenger RNA derived from term placenta directs the synthesis of a precursor to human placental lactogen (prelactogen, molecular weight 25000) in an ascites cell-free system containing ribosome free supernatant and preincubated purified ribosomes. The processing of prelactogen to native lactogen (molecular weight 22200) was only observed when a microsomal membrane preparation was added prior to the synthesis of complete protein, i.e. before release. Placental mRNA directed the synthesis of prelactogen in asystem containing free polysomes, whereas in a comparable system containing membrane-bound polysomes both prelactogen and lactogen were synthesized. The prelactogen synthesized in the latter system could be cleaved by the addition of membranes at the start of incubation. Preprotein cleavage activity was inhibited 100% by 0.04% Tritonx-100, while protein synthesis was inhibited only about 30%. Using Triton to block cleavage specifically at intervals after mRNA and membrane additions, it was determined that the overall cleavage reaction required about 15 min. When the ascites system was incubated with charged initiator [35S]methionyl-tRNAfMet prelactogen was formed. The labeled prelactogen was processed when membranes were added after 60 min of incubation. The results indicate that prelactogen is the primary gene product, and cleavage activity is apparently associated only with the membrane-bound ribosomal fraction of the cell.