Protein Translocation into ER + Signal Recognition Particle

The Endoplasmic Reticulum membrane is a busy place like an airport. Most of the integral membrane proteins and soluble proteins are co-translationally targeted, inserted and assembled here at sites referred to as translocons which consist of a group of membrane proteins that act in concert with ribosomes and molecular chaperones to facilitate:

• the insertion of integral membrane proteins into the lipid bilayer
• the translocation of soluble proteins into the ER lumen

ER Signal Sequences

Protein targeting to the ER membrane can occur co-translationally or post-translationally depending on the hydrophobicity and location of the signal sequence (SS) which consist of:

• a short sequence of hydrophobic residues
• a charged, positive N-terminal region on one side
• an uncharged, polar C-terminal region on the other side

In the post-translational process, the synthesized proteins are targeted and inserted (or translocated) after translation by the cytosolic ribosomes.

In the co-translational process, targeting of soluble and integral membrane proteins is mediated by the conserved Signal Recognition Particle.

Signal Recognition Particle and Signal Recognition Particle-Receptor


The eukaryotic Signal Recognition Particle (SRP) is composed of:

• 300-nucleotide long 7S RNA
• six protein subunits 

It binds to a hydrophobic domain [either to an N-terminal SS or a transmembrane (TM) signal segment] in the nascent polypeptide chain as it emerges from the ribosome during synthesis. 


Protein synthesis is momentarily arrested when SRP docks the ribosome–nascent chain (RNC)–SRP complex to the ER membrane with the help of the SRP receptor (SR). The SR is a heterodimer formed by the GTPases SR-α and SR-β and interacts with both the ribosome and SRP.


Interaction between the SRP--SR requires binding of GTP to both of them. This causes conformational changes in SR. As a result, the RNC is transferred from the SRP to the Sec61 translocon. Meanwhile GTP hydrolysis also triggers dissociation of the SRP-SR complex.


Translocon Structure

This complex possesses a gating capability that is functional in two directions:

• across the ER membrane
• laterally into the lipid bilayer

The mammalian complex is composed of the Sec61 α-subunit, β-subunit, γ-subunit together with the translocating chain-associating membrane protein (TRAM).

The Sec61 complex

The eukaryotic Sec61 is a heterotrimeric membrane protein complex consisting of:

• α-subunit --> this is the main channel of the translocon complex. It crosses the membrane 10 times and both its N-terminus and C-terminus face the cytosol. In its inactive state, the diameter of the cytosolic end of the channel has a diameter of 20–25 Å narrowing down in middle of the membrane to 5 Å due to presence of a ring of bulky hydrophobic residues then widening again towards the ER lumen
• β-subunit --> this is the smallest component of the Sec61 complex consisting of only 1 transmembrane domain. Although inessential for translocation or insertion, it has known to kinetically facilitate translocation by interacting with the SR heterodimer
  
• γ-subunit --> this subunit has two helices connected by an extended loop

Translocating chain-associating membrane protein (TRAM)

TRAM is an integral membrane protein with eight transmembrane segments with both N-terminus and C-terminus facing the cytosol. TRAM is responsible for the insertion of SS (containing short hydrophobic sequences) into the membrane during translocation of soluble proteins.


TRAM is known to contain charged residues within its transmembrane segments so it is thought that it acts as a chaperone by providing a more favorable environment.


Translocon associated proteins (TRAP)



Some other membrane proteins such as the translocon-associated protein (TRAP) have been reported to interact with the translocon and modulate its function. They are known to play a role in coordination of helix-helix interactions important in insertion of multiple pass proteins. 

Mechanism of Protein Translocation in ER

During co-translational insertion and/or translocation, the nascent polypeptide is squeezed into the translocon from where it exits the ribosome (ribosome exit tunnel). 

The process starts with the interaction between the translocon complex and the ribosome (see figure).

Single Pass proteins

Transmembrane helices follow a systematic insertion pathway in  a process that is thermodynamic in nature. They travel from the ribosome exit tunnel into the Sec61 translocon channel and ultimately exit the channel laterally into the surrounding lipids. 


What factors drive the integration of proteins into the membrane?

• hydrophobicity - 
• amino acid composition and position - Leu and Ala make up the bulk
• orientation of the segment - see section on topology 
• length of the helix

The formation of an α-helix is critical for membrane insertion of a segment. Without this secondary structure, it is difficult for even the most hydrophobic polypeptides to be inserted into lipid bilayers! 

Multiple Pass Proteins


In case of multiple pass membrane proteins, several transmembrane segments in a single polypeptide need to be integrated into the membrane. 


That means the first hydrophobic segment to enter the lipid bilayer has to be relocated in order to accommodate the one that follows. Sometimes many segments are released into the bilayer in pairs or groups. This depends on the nature of the protein.


Topology


During insertion, it is essential to define:

• the number of transmembrane segments to be inserted
• the orientation of segments with respect to plane of the lipid bilayer 

Whether a segment adopts an N-teminal cytosolic or a reverse orientation depends on the following factors:

1. Folding state of the membrane domain preceding the segment - forces the segment to adopt N-terminal cytosolic orientation
2. Hydrophobicity of the segment - highly hydrophobic sequences promote N-terminal translocation
    
3. Distribution of charged residues - close to the flanking regions of the segment

Multiple pass membrane proteins tend to adopt their native orientation depending on the nature of insertion of the SS or the first segment.