Abstract
The majority of the polytopic proteins that are synthesized at the ER (endoplasmic reticulum) are integrated co-translationally via the Sec61 translocon, which provides lateral access for their hydrophobic TMs (transmembrane regions) to the phospholipid bilayer. A prolonged association between TMs of the potassium channel subunit, TASK-1 [TWIK (tandem-pore weak inwardly rectifying potassium channel)-related acid-sensitive potassium channel 1], and the Sec61 complex suggests that the ER translocon co-ordinates the folding/assembly of the TMs present in the nascent chain. The N-terminus of both TASK-1 and Kcv (potassium channel protein of chlorella virus), another potassium channel subunit of viral origin, has access to the N-glycosylation machinery located in the ER lumen, indicating that the Sec61 complex can accommodate multiple arrangements/orientations of TMs within the nascent chain, both in vitro and in vivo. Hence the ER translocon can provide the ribosome-bound nascent chain with a dynamic environment in which it can explore a range of different conformations en route to its correct transmembrane topology and final native structure.
Highlights
The majority of membrane proteins synthesized at the endoplasmic reticulum (ER) are delivered as ribosome-bound nascent chains via the SRPdependent pathway [1], and subsequently engage the Sec61 translocon responsible for the co-translational translocation of polypeptides into and across the ER membrane [1,2,3]
The most prominent cross-linking partner that could be identified in this fashion was the Sec61α subunit (Figure 1B), we observed a trimeric adduct of TWIK-related acid-sensitive potassium channel 1 (TASK-1)–Sec61α–Sec61β when the cysteine probe was located at residue 28 (Figure 1B, A28C panel)
Adduct formation depended on a stable ribosome-bound nascent chain, puromycin treatment before BMH addition abolished cross-linking to components of the ER translocon (Supplementary Figure S2 at http://www.biochemj.org/bj/456/bj4560297add.htm)
Summary
The majority of membrane proteins synthesized at the ER (endoplasmic reticulum) are delivered as ribosome-bound nascent chains via the SRP (signal recognition particle)dependent pathway [1], and subsequently engage the Sec translocon responsible for the co-translational translocation of polypeptides into and across the ER membrane [1,2,3]. In the case of polytopic, or multi-spanning, membrane proteins, several TMs must be membrane-integrated and assembled, and both the ribosome and the Sec complex play key roles in this process [6]. It is generally accepted that ER translocon-associated components, such as TRAM (translocating chain-associated membrane protein) and the TRAP (translocon-associated protein) complex, may facilitate membrane insertion [3,7,8]. The ER translocon is not a passive conduit providing access for TMs to the bilayer, but rather it actively contributes to polytopic membrane protein biogenesis by facilitating the assembly of TMs before their complete integration [8,9,10,11,12,13,14]. The association of TMs into pairs and bundles is a well-defined stage during theoretical and experimental studies of polytopic membrane protein folding [15,16], and current models suggest that this process can occur within the context of the ER translocon, where cohorts of TMs may assemble before integration [17,18,19]
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