Membrane protein insertion at the endoplasmic reticulum - PubMed (original) (raw)

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Membrane protein insertion at the endoplasmic reticulum

Sichen Shao et al. Annu Rev Cell Dev Biol. 2011.

Abstract

Integral membrane proteins of the cell surface and most intracellular compartments of eukaryotic cells are assembled at the endoplasmic reticulum. Two highly conserved and parallel pathways mediate membrane protein targeting to and insertion into this organelle. The classical cotranslational pathway, utilized by most membrane proteins, involves targeting by the signal recognition particle followed by insertion via the Sec61 translocon. A more specialized posttranslational pathway, employed by many tail-anchored membrane proteins, is composed of entirely different factors centered around a cytosolic ATPase termed TRC40 or Get3. Both of these pathways overcome the same biophysical challenges of ferrying hydrophobic cargo through an aqueous milieu, selectively delivering it to one among several intracellular membranes and asymmetrically integrating its transmembrane domain(s) into the lipid bilayer. Here, we review the conceptual and mechanistic themes underlying these core membrane protein insertion pathways, the complexities that challenge our understanding, and future directions to overcome these obstacles.

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Figures

Figure 1

Figure 1

The structural diversity of integral membrane proteins. (a) Several types of integral membrane proteins are shown in different topologies. Most types of membrane proteins are inserted by a cotranslational pathway, although some use a posttranslational pathway. ER, endoplasmic reticulum. (b) Hydrophilicity plot of a G protein–coupled receptor (bovine rhodopsin) using the Kyte-Doolittle scale. The hydrophobic regions are below the axis and indicated in gold. Note that six of the seven transmembrane domains (TMDs) are easily recognizable by their hydrophobicity, but the seventh is more polar. Sequences of the first and last TMDs are shown; polar residues within each TMD are indicated by asterisks. (c) Three-dimensional structure of bovine rhodopsin showing that TMDs are highly variable in structure; some are tilted or kinked. TMD1 and TMD7 are shown in blue and red, respectively. The unusual sequence features of TMD7 (see panel b) correspond to its unusual structure in the membrane.

Figure 2

Figure 2

Assay systems used to probe sequence determinants of insertion and topology. (a) An integral membrane protein (IMP) is targeted to and docked at the translocon via earlier transmembrane domains (gray), and a later test sequence (red) is measured for insertion. The relative amounts of the two possible outcomes are assayed, typically by a modification such as glycosylation. (b) A single-spanning IMP and its flanking sequences are systematically varied, and the substrate is presented to the translocation apparatus. The final topology achieved by this combination targeting-translocation-insertion system is assayed, again typically using glycosylation as a readout.

Figure 3

Figure 3

Basic steps in cotranslational membrane protein insertion. (a) An integral membrane protein (IMP) is recognized via a hydrophobic transmembrane domain (TMD) (or signal sequence) by the signal recognition particle (SRP) on the ribosome. This is targeted to the endoplasmic reticulum (ER) membrane via the SRP receptor (SR) and transferred to the Sec61 translocation channel. The transfer reaction is accompanied by a second recognition step, this time by the Sec61 channel. The TMD is then integrated into the membrane via lateral access to the lipid bilayer provided by the Sec61 channel. Below the last two diagrams are schematic representations of an early and late TMD integration step in which the TMD (red ) is leaving the Sec61 channel via a lateral opening. (b) Schematic diagram of the SRP positioned on the ribosome, taken from cryo-electron microscopy analyses. The signal sequence recognition domain of SRP54 (the M-domain) is indicated in green and is positioned adjacent to the exit tunnel on the ribosome. The portion of the Alu-domain that binds near the elongation factor site to slow translation is shown in pink. (c) The M-domain of SRP54 in two views with hydrophobic residues indicated in green. The red sphere represents the site of signal sequence (or TMD) binding within the hydrophobic groove.

Figure 4

Figure 4

The Sec61 translocon and potential accessory factors. (a) Structural representation of the archaeal homolog of the Sec61 complex as viewed from the endoplasmic reticulum (ER) lumen and from the plane of the membrane (rotated by 90°). The left and right halves show a pseudo twofold symmetry. The helices are organized around a central pore that is occluded by a short plug helix (purple). The two blue helices are helix 2 and helix 7, which form the so-called lateral gate. When these are separated, they provide a route of access to the lipid bilayer from the central pore. In the lipid bilayer view, note that the plug (which is in the central pore) is directly behind the lateral gate, illustrating how lipid access could be mediated when the gate helices move apart. (b) Schematic representation of the structures in panel a. (c) Schematic representation of how lateral exit of a transmembrane domain (TMD) from the Sec61 channel (as viewed from the ER lumen) could be influenced by _trans_-acting accessory factors. The baseline model shows the TMD (red ) tethered to a nascent chain (black line). The nascent chain is in the central channel, whereas the TMD is at the lateral gate. The arrows above the diagram indicate the partitioning of the TMD between the lateral gate and surrounding lipid bilayer ( gray plane). This partitioning could be biased toward the membrane if the local environment was altered by either a different lipid composition or a nearby protein. Conversely, a protein that binds to the translocon could influence its dynamic properties, such as opening of the lateral gate, thereby biasing against partitioning into the lipid bilayer. In the last diagram, an accessory protein such as TRAM (translocating-chain associating membrane protein) that directly binds to a TMD could also bias movement of the TMD out of the Sec61 channel.

Figure 5

Figure 5

Models of transmembrane domain (TMD) insertion. (a) Generic model for TMD-translocon interactions. Upon its initial emergence from the ribosome, a TMD (red) enters the cytosolic vestibule of the Sec61 channel (➀). This initial metastable state allows the nascent chain to sample either of two orientations (➁, ➂). These states are envisioned to be interconvertible for a limited period of time, until the TMD laterally moves into the lipid bilayer in one or the other orientation (➃, ➄). (b) Depending on any of various parameters, the sequence of events in panel a can be biased toward one or the other outcome. In the example shown, the N-terminal domain is lengthy and partially folded (orange) by the time the TMD emerges from the ribosome. This strongly disfavors its translocation into the lumen, a requirement for sampling the type I topology. Consequently, its sampling of and eventual insertion in the type II orientation is the favored outcome. (c) An example of nonsequential insertion of TMDs in a polytopic integral membrane protein (IMP). Starting from the left, a polytopic IMP begins insertion. At the stage shown, TMD1 (yellow) has already inserted, and TMD2 (green) is entering the translocation channel. However, owing to sequence features of TMD2, it is not recognized by the translocon and is skipped, resulting in its translocation into the endoplasmic reticulum (ER) lumen. TMD3 (red) is then made and interacts with the translocon in a manner similar to that shown in panel a. Its local sequence features heavily favor the type II orientation (same as TMD1), thereby forcing segments of the previously translocated part of the protein (including TMD2) back into the translocation channel. The marginally hydrophobic TMD2 can now insert into the membrane accompanied by TMD3.

Figure 6

Figure 6

Tail-anchored (TA) protein insertion. (a) Schematic model of the known components and steps mediating posttranslational insertion of a TA protein. When the transmembrane domain (TMD) of a TA protein is synthesized, it favors recruitment of a pretargeting factor to the ribosomal surface. This is composed of Bag6, TRC35, and Ubl4A in mammals. The analogous complex in yeast is formed by Sgt2, Get4, and Get5 as well as other chaperones. Its location near the ribosome would favor capture of the TA protein upon its release. The pretargeting factor together with the targeting factor (TRC40 in mammals, Get3 in yeast) ( pink) form the TRC. This is thought to be a transient complex that facilitates sorting, recognition, and loading of the TA protein onto the targeting factor. The targeting factor is an ATPase, and its substrate-bound form is thought to be ATP-bound (indicated by a T). This is delivered to the endoplasmic reticulum (ER) membrane via a receptor composed of Get1 and Get2 in yeast (a mammalian homolog of Get1 may be WRB). The docking complex of Get1-2-3 somehow facilitates substrate release and insertion in a step that depends on ATP hydrolysis by Get3. The now-vacant Get3 (which is in a different open conformation) is recycled to the cytosol to complete the insertion cycle. (b) Structural representations of the Get3 dimer in the open conformation (lacking nucleotide) and closed conformation (with bound ADP-AlF4). In the left two structures, hydrophobic residues are shown in green, illustrating that the closed conformation contains a large hydrophobic groove. The right panel shows a hypothetical model for the closed conformation bound to the TMD region of a model TA protein. The hydrophobic TMD (19 residues) is shown in red, with flanking sequences in gold.

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