Inhibition of preprotein processing in ascites tumor lysates by incorporation of a leucine analog

Abstract

Leucine analogs were tested in the Krebs II [mouse] ascites cell-free translation system for the ability to inhibit preprotein cleavage by replacing leucine in nascent chains of bovine preprolactin, rat preprolactin, human placental prelactogen (pre-hPL), and pre-.alpha. subunit of human chorionic gonadotropin (.alpha.-hCG). In the absence of analog, ascites microsomal membranes cleaved these preproteins to their mature forms and sequestered the processed products. Two asparagine residues in .alpha.-hCG were glycosylated. When 4 mM .beta.-DL-hydroxyleucine was added to the lysate instead of L-leucine, cotranslational processing and sequestration of both species of preprolactin and pre-hPL were inhibited. Sequential Edman degradation confirmed that pre-hPL was not cleaved. The inhibition of processing by .beta.-hydroxyleucine resulted from its incorporation into protein. This was shown by reversal of the effect by addition of leucine and by inhibition of [3H]leucine incorporation into protein. Of significance, the processing of pre-.alpha.-hCG was less sensitive to .beta.-hydroxyleucine because its prepeptide contains only 4 scattered leucine residues, whereas the presegments of hPL and the prolactins contain 6-8 clustered leucine residues. Translocation and processing of secretory proteins require structural features determined by the primary amino acid sequence.