Use of synthetic signal sequences to explore the protein export machinery

Abstract

The information for correct localization of newly synthesized proteins in both prokaryotes and eukaryotes resides in self‐contained, often transportable targeting sequences. Of these, signal sequences specify that a protein should be secreted from a cell or incorporated into the cytoplasmic membrane. A central puzzle is presented by the lack of primary structural homology among signal sequences, although they share common features in their sequences. Synthetic signal peptides have enabled a wide range of studies of how these “zipcodes” for protein secretion are decoded and used to target proteins to the protein machinery that facilitates their translocation across and integration into membranes. We review research on how the information in signal sequences enables their passenger proteins to be correctly and efficiently localized. Synthetic signal peptides have made possible binding and crosslinking studies to explore how selectivity is achieved in recognition by the signal sequence‐binding receptors, signal recognition particle, or SRP, which functions in all organisms, and SecA, which functions in prokaryotes and some organelles of prokaryotic origins. While progress has been made, the absence of atomic resolution structures for complexes of signal peptides and their receptors has definitely left many questions to be answered in the future. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 307–319, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Engineering Anti-vascular Endothelial Growth Factor Single Chain Disulfide-stabilized Antibody Variable Fragments (sc-dsFv) with Phage-displayed sc-dsFv Libraries

“…Because the disulfide bonds of the newly synthesized preprotein can only be formed in the oxidizing environment of periplasm, the mechanism for the translocation of the nascent unfolded polypeptide chain from the translation site in the cytoplasm across the periplasm membrane could be a key determinant for the folding and, consequently, for the expression of the displayed protein on the phage surface (25)(26)(27)(28)(29)(30)(31). Alternative sequences in the signal peptide region have been known to modulate the expression level and folding quality of the displayed protein (25,27,31), but difficulty remains in identifying optimum signal sequences in a vast sequence space for some sc-dsFv constructs.…”

“…The key discovery is that the signal sequence variants of the sc-dsFv-pIII fusion protein have varying effects in directing the sc-dsFv expression on the recombinant phage surface. The signal sequence has been known to be responsible for the Sec system-dependent translocation of the pIII fusion protein from the translation site in the cytoplasm to the periplasm membrane (25)(26)(27)(28)(29)(30)(31), a critical process for the integration of the displayed protein on the recombinant phage surface (32). But the optimal signal sequences for the translocation of the pIII fusion protein were not known.…”

Chimeras taking shape: Potential functions of proteins encoded by chimeric RNA transcripts

“…The 1999 Nobel Prize in Physiology or Medicine was awarded to Gunter Blobel for the discovery that proteins have intrinsic signals that govern their transport and localization within the cell (Emanuelsson et al 2007;Clérico et al 2008). Indeed, these signal sequences were shown to serve as zipcodes, specifying the eventual destination of the proteins.…”

“…Signal peptides targeting proteins to the endoplasmic reticulum (ER) membrane in eukaryotes are 15-30 amino acids long, self-contained and removed after targeting (Emanuelsson et al 2007;Clérico et al 2008). In eukaryotes, proteins translocated across the ER membrane are by default transported through the Golgi apparatus and then exported by secretory vesicles (Emanuelsson et al 2007;Clérico et al 2008). Some chimeras incorporate signal peptides that could direct proteins to the ER and Golgi apparatus.…”

A career pathway in protein folding: From model peptides to postreductionist protein science

“…23 We found that functional signal sequences were required to have some welldefined properties: an ability of the hydrophobic core to fold as an a-helix, a detergent-like tendency to interact favorably with a membrane, but an inability to stably incorporate into a bilayer as a transmembrane helix. 20,[24][25][26][27][28][29][30][31] In subsequent work, we and others have been able to reconcile how these essential properties allow the secretory machinery to specifically and productively bind and manage signal peptides.…”