protein targeting

Protein targeting and the role of SRP.

Proteins are integrated into the ER membrane by at least three routes and these are perhaps best characterised in yeast. A&B) the co-translational signal recognition particle (SRP)-dependent and post-translational SRP-independent routes both of which require an amino-terminal signal sequence on the protein being targeted. These two pathways, which have preferences for more (A) or less (B) hydrophobic signal sequences converge on a common, evolutionarily conserved translocation apparatus (the TRANSLOCON) composed of the Sec61p, Sbh1p and Sss1p proteins. Also important for both of these routes are the Sec63p and Kar2p proteins. The post-translational route additionally requires the Sec62p complex composed of Sec62p 71p and 72p which appear to play a role in substrate recognition. A second translocon has been characterised in yeast, which is composed of Ssh1p (a homologue of Sec61p), Sbh2p (a homologue of Sbh1p) and Sss1p. This translocon is dedicated to the co-translational pathway. C) the post-translational insertion of C-terminally tail-anchored proteins. This pathway is less well characterised. It has been suggested that SRP plays a role in this pathway - but this is controvertial.

SRP is an evolutionarily conserved ribonucleoprotein containing, in eukaryotes, the functions of signal sequence and ribosome binding, as well as the ability to slow translation rate by the ribosome. This activity lies in the "alu" domain of the particle comprising the 5' and 3' ends of the SRP RNAand containing the SRP9 and SRP14 proteins. We are particularly interested in determining how this activity of SRP is achieved and the interactions required for it. Most of the work carried out in the laboratory uses the yeast Saccharomyces cerevisiae as the experimental system. In this organism the SRP RNA is atypically large (522 bases cf ~300 in most other eukaryotes). Recent projects have included a determination of the secondary structure of the S. cerevisiae SRP RNA secondary structure [ref][ref.]. We found that much of the "extra" RNA is in "expansions" to the Alu-domain, while the remainder of the RNA is relatively conserved and similar to other SRP RNAs. The model below is the current "working model", and is the result of continued efforts to improve the first "working model" through analysing the phenotypes of mutants, incorporation of further new SRP RNA sequences into phylogenetic comparisons and a collaborative effort with the laboratories of Dr. Tore Samuelsson and Dr. Christian Zwieb to generate consensus models and a consistent nomenclature for SRP RNAs [ref.].

Current projects include a determination of the organisation of the Alu-domain of the yeast SRP. This contains 2 copies of Srp14p, homologous to the mammalian SRP14 protein, and a single Srp21p, which comprises a domain homologous to mammalian SRP9 and a separate C-terminal domain. Mammalian SRP contains a heterodimer of SRP9+SRP14 and thus the yeast complex is significantly different at the protein level. In a second project we are dissecting the need for flexibility within the SRP RNA.