Control of Translocation through the Sec61 Translocon by Nascent Polypeptide Structure within the Ribosome

Abstract

During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocross-linking probes to detect the proximity of ribosomebound nascent polypeptides to Sec61␣. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs. TM2 first contacted Sec61␣ when the probe was positioned ϳ38 residues from the ribosome peptidyltransferase center, and TM2-Sec61␣ photoadducts decreased markedly when the probe was >80 residues from the peptidyltransferase center. Unexpectedly, as nascent chain length was gradually extended, photocross-linking at multiple sites within TM2 abruptly and transiently decreased, indicating that TM2 initially entered, withdrew, and then re-entered Sec61␣. This brief reduction in TM2 photocross-linking coincided with TM3 synthesis. Replacement of TM3 with a secretory reporter domain or introduction of proline residues into TM3 changed the TM2 cross-linking profile and this biphasic behavior. These findings demonstrate that the primary and likely secondary structure of the nascent polypeptide within the ribosome exit tunnel can influence the timing with which topogenic determinants contact, enter, and pass through the translocon. Protein translocation into the endoplasmic reticulum (ER)2 is initiated when a signal sequence emerges from the ribosome, binds a signal recognition particle, and targets the ribosomenascent chain complex to the Sec61 translocon (1-3). As the signal sequence engages Sec61␣, the nascent polypeptide is directed through an aqueous channel that extends from the exit tunnel of the 60 S ribosome subunit, through the translocon pore and into the ER lumen (4 -7). Secretory and transmembrane proteins usually traverse this pathway coincident with protein synthesis and contact translocon components (e.g. Sec61␣) when the nascent chain has extended Ͼ30 residues beyond the ribosome peptidyltransferase center (PTC) (8 -11). However, polypeptide elongation does not provide the sole driving force for ribosome-dependent translocation because, under certain circumstances, vectoral transport can be uncoupled from protein synthesis. Unfolded, ribosome-attached protein domains can be efficiently transported into the ER lumen after synthesis (12-15), and ribosome-attached polypeptides can move from the ER lumen back into the cytosol (16,17). Net anterograde movement is therefore controlled by several factors, including polypeptide folding, attachment of N-linked glycans, interaction with ER chaperones, and ultimately, release of the nascent polypeptide following peptidyl-tRNA bond cleavage (16, 18 -20).In contrast to events in the ER lumen, relatively little is known regarding how vectoral movement of the nascent polypeptide is controlled within th...

A signal-anchor sequence stimulates signal recognition particle binding to ribosomes from inside the exit tunnel

“…It is attractive to speculate that in vivo a preference of SRP for its prime substrates is facilitated by recruitment to RNCs translating hydrophobic SAs even before these emerge at the tunnel exit. Although the mechanism is not understood, it may resemble ribosomal recognition of a nascent transmembrane domain that was found to affect the operational mode of the ribosome-bound translocon in the ER membrane (30,35,36). Consistent with such a model, it is the hydrophobicity of a signal sequence that determines whether or not a protein enters the coor posttranslational pathway in vivo (16).…”

Dissecting the physiological role of selective transmembrane-segment retention at the ER translocon

“…S4). Although crosslinking using BMH, or other such reagents, can never achieve complete efficiency, the synchronisation of stalled nascent chains and the analysis of only C-terminally tagged polypeptides ensures that this approach accurately reports the environment of the bulk of the P2X2 chains present in each reaction (see also Daniel et al, 2008;Ismail et al, 2006;Mothes et al, 1997;Pitonzo et al, 2009). …”

Lateral opening of a translocon upon entry of protein suggests the mechanism of insertion into membranes

“…Assignment of topology is partially determined by electrostatic interaction between charged residues flanking the signal core and charged residues located at the plug and the C-terminal end of TM8 in the channel; these residues are likely to contribute to an electrostatic clamp. The study of the Sec61-mediated biogenesis of aquaporin-4, an all α-helical integral polytopic protein (37,38), showed that each TM interacted with and moved through Sec61α in a highly ordered and sequential manner and suggested the presence of a single primary binding site. Our structure may reveal the modality of such interactions at this primary binding site.…”