Question 1

How do multipass transmembrane proteins get inserted into the ER membrane?

Accepted Answer

Multipass transmembrane proteins are inserted through the Sec61 translocon channel during translation. The N-terminal signal sequence initiates translocation, and when hydrophobic domains are encountered, the translocon pauses and opens its lateral gate to embed each domain into the lipid bilayer. Translation resumes between domains, creating alternating transmembrane and cytosolic regions until the protein is complete.

Question 2

What determines the orientation of N and C termini in multipass transmembrane proteins?

Accepted Answer

The number of transmembrane domains determines terminal orientation. Proteins with odd-numbered transmembrane domains position their N and C termini on opposite sides of the membrane, while those with even-numbered domains align both termini on the same side. Additionally, the positioning of positive residues before or after the first transmembrane domain influences whether the N-terminal faces the cytosol or ER lumen.

Question 3

What role does the signal recognition particle play in multipass protein insertion?

Accepted Answer

The signal recognition particle (SRP) and its receptor (SR) initiate translocation of the first transmembrane domain through the ER membrane. However, their involvement is limited to the initial domain insertion. Threading of subsequent transmembrane domains is independent of the SRP-SR complex and depends primarily on the hydrophobicity of each translated domain and the ribosome-translocon assembly.

Question 4

How do accessory complexes assist in multipass protein insertion?

Accepted Answer

Accessory complexes like TRAP and TRAM interact with the Sec61 translocon to facilitate accurate folding, insertion, and assembly of multipass transmembrane proteins. While the Sec61 translocon-ribosome assembly forms the core of protein translocation, these accessory complexes are essential because multipass proteins have complex biophysical features that require additional support for proper membrane integration.

Question 5

What factors influence the topology of multipass transmembrane proteins?

Accepted Answer

Multiple factors affect multipass protein topology, including the length and hydrophobic profile of transmembrane domains, the distance between consecutive domains, and the length of extra-membrane loops. These biophysical characteristics determine how each domain interacts with the translocon and lipid bilayer, ultimately establishing the protein's final spatial arrangement and orientation within the ER membrane.

Question 6

Why is multipass protein insertion more complex than single-pass protein insertion?

Accepted Answer

Multipass protein insertion is more complex because it requires multiple cycles of translocation pausing and resumption for each hydrophobic domain. Unlike single-pass proteins, multipass proteins must coordinate the sequential embedding of multiple transmembrane domains while maintaining proper topology. The interplay of domain hydrophobicity, loop lengths, and accessory protein complexes makes their insertion significantly more challenging than insertion of single-pass transmembrane proteins.

Question 7

What happens to the signal sequence during multipass protein insertion?

Accepted Answer

The N-terminal ER signal sequence acts as the start-transfer cue for initial polypeptide translocation down the Sec61 channel. Unlike single-pass proteins, multipass proteins typically lack a cleavable signal sequence and instead use their first hydrophobic transmembrane domain as the ER signal sequence. The signal peptidase complex cleaves this initial sequence on the ER membrane after translocation begins.

Inserimento di proteine transmembrana multi-passaggio nel RER

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