Evolutionarily conserved binding of ribosomes to the translocation channel via the large ribosomal RNA

Abstract

During early stages of cotranslational protein translocation across the endoplasmic reticulum (ER) membrane the ribosome is targeted to the heterotrimeric Sec61p complex, the major component of the proteinconducting channel. We demonstrate that this interaction is mediated by the 28S rRNA of the eukaryotic large ribosomal subunit. Bacterial ribosomes also bind via their 23S rRNA to the bacterial homolog of the Sec61p complex, the SecYEG complex. Eukaryotic ribosomes bind to the SecYEG complex, and prokaryotic ribosomes to the Sec61p complex. These data indicate that rRNA-mediated interaction of ribosomes with the translocation channel occurred early in evolution and has been conserved. Keywords: protein translocation/ribosome/rRNA/SecY/ Sec61 IntroductionProtein translocation across the ER membrane can occur either co-or post-translationally. In the cotranslational pathway, a polypeptide chain is synthesized on a membrane-bound ribosome; the elongating chain is transferred from a tunnel in the ribosome through a channel in the membrane into the lumen of the ER (Crowley et al., 1994;Mothes et al., 1994;Beckmann et al., 1997). In the posttranslational pathway, the chain is ®rst completed in the cytosol before being transported across the membrane. In both cases the same membrane channel is used, formed by three or four copies of the evolutionarily conserved heterotrimeric Sec61p complex. In the cotranslational pathway, the Sec61p channel associates with the ribosome (Go Èrlich and Rapoport, 1993;Kalies et al., 1994;Hanein et al., 1996;Beckmann et al., 1997), and in the posttranslational pathway, at least in the yeast Saccharomyces cerevisiae, it associates with the tetrameric Sec62/63p membrane protein complex to form a heptameric Sec complex (Deshaies et al., 1991;Panzner et al., 1995;Hanein et al., 1996).How the ribosome binds to the Sec61p complex during cotranslational translocation is unknown, and it is unclear whether the interaction involves ribosomal RNA (rRNA), protein or both. Non-translating mammalian ribosomes can bind with high af®nity to the Sec61p complex in mammalian ER membranes (Rolleston, 1972;Borgese et al., 1974;Kalies et al., 1994). This interaction is salt sensitive, and the binding site is likely to be located in the large ribosomal subunit (Rolleston, 1972;Borgese et al., 1974;Unwin, 1977). The association of non-translating ribosomes with the Sec61p complex mimics early stages of cotranslational protein translocation (Jungnickel and Rapoport, 1995;Beckmann et al., 1997;Neuhof et al., 1998). When the signal sequence of a growing nascent chain has just emerged from the ribosome, the ribosome± nascent chain complex is targeted by the signal recognition particle (SRP) to the membrane (Walter and Blobel, 1981) and initially binds to the Sec61p complex in a saltsensitive manner (Jungnickel and Rapoport, 1995). After further chain elongation, the signal sequence is recognized by the channel and inserts into it, and the ribosome± membrane interaction becomes salt resistant (Jungnickel and Rapopor...

Ribosome Binding to and Dissociation from Translocation Sites of the Endoplasmic Reticulum Membrane

“…To test whether the dissociation rate of nontranslating ribosomes is consistent with such a model, we labeled ribosomes with a reagent that attaches 35 S to amino groups. These ribosomes sedimented at the expected 80S position in a sucrose gradient (Supplemental Figure 2) and bound to PK-RMs with a binding constant of 50 nM, consistent with previous results (our unpublished data; Prinz et al, 2000a). To test ribosome dissociation from the membrane, radiolabeled ribosomes were incubated with PK-RMs, the samples were diluted to prevent rebinding, and the membranes were sedimented at different times.…”

“…Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al, 1974;Kalies et al, 1994;Prinz et al, 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al, 1973) and do not easily dissociate from the membrane (Borgese et al, 1973;Potter and Nicchitta, 2002).…”

“…In membrane proteins, TM sequences are partitioned sideways through the walls of the channel into the lipid phase, leaving some parts of the polypeptide chain in the cytosol and others in the ER lumen. For both classes of proteins, the translating ribosome stays bound to the channel during the entire translocation process (Mothes et al, 1997).Previous work has shown that the Sec61 complex can bind not only RNCs but also nontranslating ribosomes with nanomolar affinity (Borgese et al, 1974;Kalies et al, 1994;Prinz et al, 2000a). Ribosomes remain bound to ER membranes after nascent chain release (Adelman et al, 1973) and do not easily dissociate from the membrane (Borgese et al, 1973;Potter and Nicchitta, 2002).…”

The α and β Subunit of the Nascent Polypeptide-associated Complex Have Distinct Functions

“…This hypothesis is supported by the observation that RNase A treatment of ribosomes results in less holoNAC binding to ribosomes. 3 Interestingly, evidence has recently been provided that 28 S rRNA mediates the interaction of the ribosome with the SEC61 complex (38), which together with NAC and SRP, has been shown to compete for binding to the M-site (23). Also consistent with this idea is our unpublished finding that both tRNA and mRNA compete for the cross-link of ␣NAC to the nascent chain but have no effect on the cross-link of holoNAC to nascent chains.…”

Two-stage Binding of SecA to the Bacterial Translocon Regulates Ribosome-Translocon Interaction

“…Studies with proteoliposomes indicated that no additional components were necessary for this interaction, since purified SecYEG protein was sufficient to restore high affinity translocon-binding activity for both SecA and ribosomes (23,24). In the case of SecA, this activity resides within its N-domain, which is homologous to DEAD helicase motor domains and consists of two nucleotide-binding domains that flank a substrate protein-binding site (25)(26)(27)(28).…”