Abstract
Polytopic protein topology is established in the endoplasmic reticulum (ER) by sequence determinants encoded throughout the nascent polypeptide. Here we characterize 12 topogenic determinants in the cystic fibrosis transmembrane conductance regulator, and identify a novel mechanism by which a charged residue is positioned within the plane of the lipid bilayer. During cystic fibrosis transmembrane conductance regulator biogenesis, topology of the C-terminal transmembrane domain (TMs 7-12) is directed by alternating signal (TMs 7, 9, and 11) and stop transfer (TMs 8, 10, and 12) sequences. Unlike conventional stop transfer sequences, however, TM8 is unable to independently terminate translocation due to the presence of a single charged residue, Asp(924), within the TM segment. Instead, TM8 stop transfer activity is specifically dependent on TM7, which functions both to initiate translocation and to compensate for the charged residue within TM8. Moreover, even in the presence of TM7, the N terminus of TM8 extends significantly into the ER lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis.
Highlights
Even in the presence of TM7, the N terminus of TM8 extends significantly into the endoplasmic reticulum (ER) lumen, suggesting a high degree of flexibility in establishing TM8 transmembrane boundaries. These studies demonstrate that signal sequences can markedly influence stop transfer behavior and indicate that ER translocation machinery simultaneously integrates information from multiple topogenic determinants as they are presented in rapid succession during polytopic protein biogenesis
To compare the behavior of topogenic determinants encoded within CFTR TMD1 and TMD2, coding sequences were truncated after each TM segment and tagged at the C terminus with a translocation reporter derived from bovine prolactin
We fused a C-terminal translocation reporter after each TM segment of CFTR to identify topogenic determinants responsible for generating the 12spanning topology characteristic of many mammalian ABC transporters. These results provide a framework for describing the sequence of translocation initiation and termination events that give rise to CFTR transmembrane topology and identify a novel mechanism of cooperation between TM segments during early biogenesis events
Summary
The polypeptide into the lipid bilayer (reviewed in Refs. 1–3). In the simplest model, topology can be established cotranslationally by alternating topogenic determinants that function as signal (anchor) and stop transfer sequences (4 –7). TM7 was required for TM8 stop transfer activity because TM8 failed to terminate translocation when engineered into an otherwise secretory protein containing a cleaved signal sequence These studies suggest that in addition to functioning as a signal sequence, TM7 enables ER translocation machinery to properly recognize TM8 as a transmembrane segment positioning D924V in the plane of the membrane. They provide evidence that stop transfer activity can be regulated in a novel and complex manner as multiple transmembrane segments are presented to the translocon in rapid succession during polytopic protein biogenesis
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