Signal Recognition Particle Alu Domain Occupies a Defined Site at the Ribosomal Subunit Interface upon Signal Sequence Recognition

Abstract

The eukaryotic signal recognition particle (SRP) is essential for cotranslational targeting of proteins to the endoplasmic reticulum (ER). The SRP Alu domain is specifically required for delaying nascent chain elongation upon signal sequence recognition by SRP and was therefore proposed to interact directly with ribosomes. Using protein cross-linking, we provide experimental evidence that the Alu binding protein SRP14 is in close physical proximity of several ribosomal proteins in functional complexes. Crosslinking occurs even in the absence of a signal sequence in the nascent chain demonstrating that SRP can bind to all translating ribosomes and that close contacts between the Alu domain and the ribosome are independent of elongation arrest activity. Without a signal sequence, SRP14 cross-links predominantly to a protein of the large subunit. Upon signal sequence recognition, certain cross-linked products become detectable or more abundant revealing a change in the Alu domain-ribosome interface. At this stage, the Alu domain of SRP is located at the ribosomal subunit interface since SRP14 can be cross-linked to proteins from the large and small ribosomal subunits. Hence, these studies reveal differential modes of SRP-ribosome interactions mediated by the Alu domain.Ribosomes translating mRNAs coding for secretory and membrane proteins are specifically targeted to the endoplasmic reticulum (ER) membrane by a cytosolic ribonucleoprotein particle, the signal recognition particle (SRP) (for review, see ref 1). The specificity of this process is ensured by the presence of a signal sequence in the growing peptide chain, which is recognized by SRP when it emerges from the ribosome. Signal sequence recognition by SRP causes a slow down or an arrest in the elongation of the nascent chain (2, 3). The SRP-ribosome-nascent chain complex is then targeted to the ER via the interaction of SRP with its membrane receptor, the SRP receptor (SR), a heterodimeric membrane protein (4,5). SR coordinates the release of SRP from the ribosome with the insertion of the nascent chain into the Sec61 complex, the aqueous translocation pore in the ER membrane (for reviews, see refs 6 and 7). Free SRP can then engage in another targeting round, and membraneassociated ribosomes resume translation at their regular speed, leading to the cotranslational transfer of the nascent chain across or into the ER membrane. As emphasized by

Direct binding of the Alu binding protein dimer SRP9/14 to 40S ribosomal subunits promotes stress granule formation and is regulated by Alu RNA

“…Fragile X Mental Retardation Protein (FMRP) was detected using the monoclonal antibody 1C3 (30). Affinity-purified anti-human SRP14, anti-human SRP19, anti-human SRP72 and anti-S15 antibodies were described before (7,28,31). Anti-human SRP9 antibodies were raised in chicken and affinity-purified.…”

Residues in SRP9/14 essential for elongation arrest activity of the signal recognition particle define a positively charged functional domain on one side of the protein

“…Purification of the proteins was described earlier (Huck et al 2004;Terzi et al 2004). All proteins were stored in 20 mM HEPES (pH 7.5), 500 mM KOAc (pH 7.5), 0.1 mM EDTA (pH 7.5), 0.01% NIKKOL, 10 mM DTT, and 10% glycerol.…”

Access to Ribosomal Protein Rpl25p by the Signal Recognition Particle Is Required for Efficient Cotranslational Translocation

“…Cross-linking and cryo-electron microscopic (EM) studies have identified multiple contact sites between these targeting/translocation components and the ribosome. SRP makes six contacts with the ribosome: four located around the ribosomal exit site and two at the subunit interface (Pool et al, 2002;Halic et al, 2004;Terzi et al, 2004), which represent the sites involved in signal sequence recognition and elongation arrest, respectively. The Sec61p complex interacts with the ribosome at between four and six contact points, again located at the ribosomal exit site (Beckmann et al, 2001;Menetret et al, 2005).…”