Different sec-requirements for signal peptide cleavage and protein translocation in a model E. coli protein

AbstractWe describe a secretory E. coli protein with a novel phenotype: signal peptide cleavage is largely unaffected whereas chain translocation is efficiently blocked under conditions where SecA, a central component of the secretory machinery, is rendered non-functional, and we have traced this phenotype to the presence of a mildly hydrophobic segment located ~30 residues downstream of the signal peptide. When this segment is deleted, normal SecA-dependent signal peptide cleavage and chain translocation is observed; when its hydrophobicity is increased, it becomes a permanent membrane anchor with cleavage of the signal peptide and membrane insertion both being SecA-independent. These findings suggest that the initial insertion of the signal peptide across the membrane can be uncoupled from the translocation process proper

A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

We have developed a new method for identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs significantly better than previous prediction schemes, and can easily be applied on genome-wide data sets. Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, thoughwith lower precision. Predictions can be made on a publicly available WWW server. Present address: Novo Nordisk A/S, Scientific Computing, Building 9M1, Novo Alle, DK-2880 Bagsværd, Denmark Introduction Signal peptides control the entry of virtually all proteins to the secretory pathway, both in eukaryotes and prokaryotes (von Heijne, 1990; Gierasch, 1989; Rapoport, 1992). They comprise the N--terminal part of the amino acid chain, and are cleaved off while the protein is translocated through the membrane. The common structure of signal peptides from variou..

Different conformations of nascent polypeptides during translocation across the ER membrane

BACKGROUND: In eukaryotic cells, proteins are translocated across the ER membrane through a continuous ribosome-translocon channel. It is unclear to what extent proteins can fold already within the ribosome-translocon channel, and previous studies suggest that only a limited degree of folding (such as the formation of isolated α-helices) may be possible within the ribosome. RESULTS: We have previously shown that the conformation of nascent polypeptide chains in transit through the ribosome-translocon complex can be probed by measuring the number of residues required to span the distance between the ribosomal P-site and the lumenally disposed active site of the oligosaccharyl transferase enzyme (J. Biol. Chem 271: 6241-6244).Using this approach, we now show that model segments composed of residues with strong helix-forming properties in water (Ala, Leu) have a more compact conformation in the ribosome-translocon channel than model segments composed of residues with weak helix-forming potential (Val, Pro). CONCLUSIONS: The main conclusions from the work reported here are (i) that the propensity to form an extended or more compact (possibly α-helical) conformation in the ribosome-translocon channel does not depend on whether or not the model segment has stop-transfer function, but rather seems to reflect the helical propensities of the amino acids as measured in an aqueous environment, and (ii) that stop-transfer sequences may adopt a helical structure and integrate into the ER membrane at different times relative to the time of glycan addition to nearby upstream glycosylation acceptor sites

RNA splicing: Advantages of parallel processing

Parallel and sequential modes of RNA processing are systematically compared by an analysis of the relevant kinetic reaction schemes. The parallel mode is shown to be superior in the sense that it allows molecules to be processed with larger numbers of introns, smaller losses of immature intermediates, and shorter processing times. It also is more sensitive to variations in the rate constants for individual splice-reactions, and hence more amenable to evolutionary refinements. Quantitatively, the parallel mode agrees well with published experimental data.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/23829/1/0000068.pd

Biological insertion of computationally designed short transmembrane segments

The great majority of helical membrane proteins are inserted co-translationally into the ER membrane through a continuous ribosome-translocon channel. The efficiency of membrane insertion depends on transmembrane (TM) helix amino acid composition, the helix length and the position of the amino acids within the helix. In this work, we conducted a computational analysis of the composition and location of amino acids in transmembrane helices found in membrane proteins of known structure to obtain an extensive set of designed polypeptide segments with naturally occurring amino acid distributions. Then, using an in vitro translation system in the presence of biological membranes, we experimentally validated our predictions by analyzing its membrane integration capacity. Coupled with known strategies to control membrane protein topology, these findings may pave the way to de novo membrane protein design

The E. coli SRP: preferences of a targeting factor

AbstractResearch on the targeting of proteins to the cytoplasmic membrane of E. coli has mainly focused on the so-called `general secretory pathway' (GSP) which involves the Sec-proteins. Recently, evidence has been obtained for an alternative targeting pathway in E. coli which involves the signal recognition particle (SRP). The constituents of this SRP pathway in E. coli are homologous to those of the well-characterized eukaryotic SRP pathway, which is the main targeting pathway for both proteins translocated across and inserted into the endoplasmic reticulum membrane. However, until recently, no clear function could be assigned to the SRP in E. coli. New studies point to an important role of the E. coli SRP in the assembly of inner membrane proteins