A DIRECT DETERMINATION OF THE MINIMUM SIZE FOR A EUKARYOTIC SECRETORY PRECURSOR (PREPROINSULIN, TRANSLOCATION, SIGNAL SEQUENCE, ENDOPLASMIC RETICULUM, GENE FUSION)
ESKRIDGE, ETTA MARY
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Most eukaryotic secretory proteins are synthesized as precursors containing a transient amino-terminal signal peptide of 20-25 amino acids. The signal peptide mediates binding of polysomes to the endoplasmic reticulum (ER) via a co-translational interaction with signal recognition particle (SRP) and a receptor in the ER membrane. SPR interacts with nascent precursors after about 70 amino acids have polymerized, thus the prediction is that any small secretory peptide would be synthesized as a precursor with a minimum size of 70-80 amino acids. This hypothesis was tested directly by investigating the biosynthesis of preproinsulin, a precursor of 116 amino acids, and a series of preproinsulin truncations which spanned the predicted minimum size.;Segregation and processing of preproinsulin were SRP dependent and strictly co-translational in vitro. Synchronized kinetic studies showed that membrane interaction of nascent preproinsulin occurred when about 60-70 amino acids had polymerized; molecules beyond this size were incapable of initiating a functional membrane interaction. A series of cDNA clones encoding preproinsulin-like peptides spanning the predicted minimum size of 70-80 amino acids were generated. Each clone encoded the preproinsulin signal peptide and at least a portion of the mature hormone. The clones were transcribed and translated in vitro in the presence of microsomal membranes. The results indicate that peptides of 64 and 78 amino acids, but not 45 amino acids, were able to initiate membrane binding with an efficiency almost equivalent to preproinsulin. Signal cleavage of the 64 amino acid peptide was impaired, however; this may reflect inefficient translocation of this peptide through the ER bilayer.;Chimeric genes containing portions of preproinsulin cDNA and the coding sequences for the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT) were also constructed. CAT was sequestered into microsomal vesicles when attached to the preproinsulin signal peptide and B chain. The signal sequence was removed efficiently and accurately, and the fusions were glycosylated at a cryptic Asn-Gln-Thr site in the CAT molecule. These data suggest that the truncations of preproinsulin contained all the information necessary for membrane binding, translocation and processing, implying that the 45 amino acid peptide is deficient only in meeting the minimum size requirement.