Abstract
Ovalbumin, unlike other secretory proteins, is synthesized and secreted without cleavage of a hydrophobic signal peptide. Kinetic experiments were performed in a cell-free translation system to measure the minimum size of ovalbumin nascent chains required for binding of both the nascent chain and the corresponding mRNA to microsomal membranes derived from dog pancreas. Results of these experiments revealed that 50 to 60 amino acid residues are sufficient to bind ovalbumin-synthesizing polysomes to membranes in vitro. When microsomes with associated polysomes were isolated from chick oviduct, nascent ovalbumin chains longer than 50 residues were protected from proteolysis as long as the membranes remained intact, suggesting that the polypeptides were sequestered by the endoplasmic reticulum. We conclude that the functional signal for membrane translocation of ovalbumin becomes accessible when the nascent chain is 50 to 60 residues long. We speculate that the hydrophobic sequence which lies between residues 25 and 45 folds back on the preceding residues to form an amphipathic hairpin structure which is the signal element recognized by the membrane.
Highlights
When microsomeswith associated polysomes were isolated from chick oviduct, nascent ovalbumin chainslongerthan 50 residues wereprotectedfrom proteolysis as long as the membranes remained intact, suggesting that the polypeptides were sequestered by the endoplasmic reticulum.We conclude that the funca peptide which inhibits the translocation of precursor proteins across dog pancreas microsomal membranes in a cellfree translation system
We have conducted experiments to determine the location of a signal sequence in ovalbumin by measuring the shortest nascent chains that interact withmicrosomal membranes both in vivo and in vitro.The results of our studiesare atvariance tional signal for membrane translocatioonf ovalbumin with the proposal of Lingappa et al [11]and indicate that the becomes accessible when the nascent chainis 50 to 60 signal sequence for ovalbumin secretion resides within the residues long
In a synchronized cell-free translation system containing membranes, one would expect that the mRNA for a secreted protein would become associated with membranes only after the peptide sequence that is recognized by the RER membrane became exposed
Summary
Hairpinstructurewhich is the signal element recog- Preparation of mRNA and cDNA-mRNA,t, and [3H]cDNA,~. As well as membrane proteins, are synthesized as precursors containing NHz-terminal, signal peptides of 15 to prepared essentially as described by Haines et al [15] and is >90% pure as judged by sodium dodecyl sulfate-acrylamide gel analysis of the total translation products. In both eukaryotes and prokaryotes, Cell-free Translation a n d Assay for Membrane-boundmRNA-. SDS, sodium dodecyl sulfate; ov, mu, and gb, subscripts to identify nucleic acids specifying ovalbumin, ovomucoid, and globin, respectively; OV,, a tryptic peptide from ovalbumin representing residues 229 to 276.
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