Signal patch

Abstract: According to the signal hypothesis, a signal sequence, once having initiated export of a growing protein chain across the rough endoplasmic reticulum, is cleaved from the mature protein at a specific site. It has long been known that some part of the cleavage specificity resides in the last residue of the signal sequence, which invariably is one with a small, uncharged side-chain, but no further specific patterns of amino acids near the point of cleavage have been discovered so far. In this paper, some such patterns, based on a sample of 78 eukaryotic signal sequences, are presented and discussed, and a first attempt at formulating rules for the prediction of cleavage sites is made.

Abstract: Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N-terminal extension sequence, called the signal peptide, which is required for export. It is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology. In attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function. Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N-terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophobicity within a range of length limitations; the central portion adopts an alpha-helical conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side-chain amino acids in the -1 and -3 positions. This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined. The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export. The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion. In this way, nature has indeed evolved exquisitely tuned signal sequences.

Abstract: The insertion of proteins into the endoplasmic reticulum is mediated by short hydrophobic domains called signal sequences, which are usually cleaved during insertion. We previously constructed DNAs encoding vesicular stomatitis virus glycoproteins with N-terminal extensions preceding the signal sequence and showed that these extensions allowed normal signal-sequence function and cleavage in vivo. To analyze signal sequence topology during membrane insertion, we generated a point mutation that blocks cleavage of these signal sequences. After expressing these proteins in HeLa cells, we used proteolysis of microsomal membranes to determine that the N terminus of the signal sequence and the C terminus of each protein remain on the cytoplasmic side of the endoplasmic reticulum after insertion. This result indicates that the proteins were inserted in a looped configuration. Extending this finding, we were able to reverse the orientation of such a mutant protein by deleting its normal C-terminal transmembrane and cytoplasmic domains. In addition to demonstrating that a signal sequence can function as a membrane anchor, these findings show that except for the presence of a cleavage site, the cleaved signal sequence of a type I transmembrane protein is structurally and functionally equivalent to the noncleaved signal sequences of type II transmembrane proteins.