Different Transmembrane Domains Associate with Distinct Endoplasmic Reticulum Components during Membrane Integration of a Polytopic Protein

Abstract

We have been studying the insertion of the seven transmembrane domain (TM) protein opsin to gain insights into how the multiple TMs of polytopic proteins are integrated at the endoplasmic reticulum (ER). We find that the ER components associated with the first and second TMs of the nascent opsin polypeptide chain are clearly distinct. The first TM (TM1) is adjacent to the alpha and beta subunits of the Sec61 complex, and a novel component, a protein associated with the ER translocon of 10 kDa (PAT-10). The most striking characteristic of PAT-10 is that it remains adjacent to TM1 throughout the biogenesis and membrane integration of the full-length opsin polypeptide. TM2 is also found to be adjacent to Sec61alpha and Sec61beta during its membrane integration. However, TM2 does not form any adducts with PAT-10; rather, a transient association with the TRAM protein is observed. We show that the association of PAT-10 with opsin TM1 does not require the N-glycosylation of the nascent chain and occurs irrespective of the amino acid sequence and transmembrane topology of TM1. We conclude that the precise makeup of the ER membrane insertion site can be distinct for the different transmembrane domains of a polytopic protein. We find that the environment of a particular TM can be influenced by both the "stage" of nascent chain biosynthesis reached, and the TM's relative location within the polypeptide.

Cotranslational integration and initial sorting at the endoplasmic reticulum translocon of proteins destined for the inner nuclear membrane

“…On the other hand, because of variations in probe length, reactivity, location, and target atom, one must be cautious in extrapolating from a crosslink to a specific structural arrangement between two macromolecules. For example, the second TMS in opsin was chemically crosslinked to TRAM (23), but no crosslinking to TRAM was detected when photoreactive probes were positioned in the middle of the second opsin TMS in a chimeric protein (10). Thus, high-resolution experiments using multiple different probes will be necessary to assess the extent to which the INM and non-INM TMS binding sites in the translocon overlap spatially and dynamically.…”

Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon

“…In the case of polytopic membrane proteins, multiple TMs must be integrated by the Sec61 translocon and subsequently assembled into a correctly folded polypeptide. Opsin has seven TMs, and it has been generally assumed that the individual TMs are sequentially integrated into the ER membrane as they emerge from the ribosome and engage the Sec61 complex McCormick et al, 2003;Meacock et al, 2002). Such an ordered insertion of opsin TMs is also supported by in vivo studies of an archaeal equivalent, bacterioopsin (Dale et al, 2000), and by a detailed analysis of aquaporin-4 biogenesis (Sadlish et al, 2005).…”

Double-spanning Plant Viral Movement Protein Integration into theEndoplasmic Reticulum Membrane Is Signal Recognition Particle-dependent,Translocon-mediated, andConcerted

“…In addition, photocross-linking to TRAM was observed for both TM segments of p9, although many membrane protein TM segments do not photocross-link to TRAM (17)(18)(19)(20). Thus, plant viral membrane protein integration appears to utilize the translocon apparatus and associated factors of the host to achieve targeting to and integration into the ER membrane, although the viral and host processes may differ somewhat with respect to the interaction with associated components such as TRAM.…”