Both Lumenal and Cytosolic Gating of the Aqueous ER Translocon Pore Are Regulated from Inside the Ribosome during Membrane Protein Integration

Abstract

Portions of the nascent chain are exposed to the lumen, the cytosol, or neither at different stages during the cotranslational integration of a protein into the ER membrane, as shown by compartment-specific collisional quenching of fluorophores incorporated into the polypeptide. The opening or closing of each end of the aqueous translocon pore is tightly controlled and occurs in a sequence that does not compromise the membrane's permeability barrier. Surprisingly, these structural changes at the membrane are effected by the transmembrane segment in the nascent protein from inside the ribosome. Thus, the ribosome, not the translocon, first recognizes the transmembrane segment and triggers long-range structural changes at the translocon that may be involved in shifting its function from translocation to integration.

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

“…Furthermore, during ST-mediated translocation termination, the translocon channel is gated closed at the ER lumen (Hamman et al, 1998), and the nascent chain rapidly becomes exposed to the cytosol Liao et al, 1997). Based on these observations, our model proposes that when TM7b terminates translocation, TM8 emerges from the ribosome in a cytosolically accessible orientation.…”

“…Based on these observations, our model proposes that when TM7b terminates translocation, TM8 emerges from the ribosome in a cytosolically accessible orientation. Given the short connecting peptide loop between TM7b and TM8 (13 residues), however, it is likely that TM8 is actually located between the ribosome exit site and the translocon proper (Blobel and Sabatini, 1970;Liao et al, 1997). An important aspect of this process, therefore, is precisely how the weak ST activity of TM7b interacts with TM8 to influence the gated state of the translocon channel (Liao et al, 1997).…”

“…single or double spanning) is that downstream topogenic sequences would be presented to the ER membrane in very different contexts. For example, if TM7b terminated translocation, then TM8 would likely emerge from the ribosome in contact with cytosol (Liao et al, 1997). In chains where TM7b failed to terminate translocation, however, TM8 would emerge from the ribosome on an actively translocating chain (presumably moving through the Sec61 translocation channel [Borel and Simon, 1996;Laird and High, 1997] and shielded from the cytosol by the ribosome-membrane junction [Crowley et al, 1993[Crowley et al, , 1994).…”

“…Given the short connecting peptide loop between TM7b and TM8 (13 residues), however, it is likely that TM8 is actually located between the ribosome exit site and the translocon proper (Blobel and Sabatini, 1970;Liao et al, 1997). An important aspect of this process, therefore, is precisely how the weak ST activity of TM7b interacts with TM8 to influence the gated state of the translocon channel (Liao et al, 1997). It is possible that TM7b simply fails to terminate translocation in a subset of chains and that, for these chains, the translocon remains open until it is subsequently closed by TM8.…”

Glycoprotein Folding in the Endoplasmic Reticulum

“…In the early 1990s, ER lumenal proteins, in addition to cytosolic factors, were also shown to be essential for the completion of protein translocation (Nicchitta and Blobel, 1993). The opening and closing of the pore is tightly regulated: the lumenal side is sealed by BiP before the cytosolic side opens (Liao et al, 1997) (Hamman et al, 1998). This is not the last time that we will encounter BiP, a soluble ER resident protein of the hsp70 family.…”