Protein Processing I:  Targeting to the Endomembrane System

Overview of the Endomembrane System:The Endomembrane system is a complex of membrane limited compartments involved in the processing and movement of proteins. Newly synthesized proteins enter the endomembrane system through the endoplasmic reticulum. From there them move through the Golgi apparatus and into either the Secretory Pathway or the Lysosomal Pathway. We begin by looking at targeting of proteins from the cytosol to the endoplasmic reticulum.

1. Targeting to the endoplasmic reticulum.

Synopsis. 

Synthesis of proteins entering the endoplasmic reticulum is initiated on free ribosomes. 

A targeting sequence of  hydrophobic amino acids near the N- terminal end of the growing polypeptide results in the binding of the ribosome to ER membrane and in insertion of the polypeptide into the endoplasmic reticulum.

Proteins going to Golgi, endosomes, lysosomes and ER all enter the ER and don't come out again.

Two groups of proteins are targeted to the ER:

• Proteins that are completely translocated into lumen of the ER. These  are soluble proteins destined for secretion, or for the lumen of another organelle. These proteins are  never part of membranes.
• Proteins that are inserted into membranes, and hence are only partially translocated into the endoplasmic reticuluum. These proteins may be destined for ER, another organelle, or the plasma membrane. In all of these cases the proteins stay within the membrane (e.g. cellulose synthase).

Let's deal first with the case of proteins that will be inserted into the ER lumen:

• The signal sequence is a long sequence of about 20 hydrophobic amino acid residues that contains a hydrophobic membrane crossing domain at the N terminal end.
• The emerging signal sequence combines with a 'signal recognition particle' (SRP). This greatly reduces the rate of translocation and allows the ribosome to attach to the endoplasm reticulum by means of a special SRP receptor in the ER membrane.
• The ribosome becomes attached to a ribosome receptor that also functions as the translocation channel for the newly synthesized polypeptide. As the ribosome becomes attached, the SRP is removed and translation resumes.
• The protein is fed through the translocation channel.
• After translation is complete, the signal sequence, which is embedded into the ER membrane, is cleaved off of the protein by a specific signal peptidase, an enzyme that is present in the ER lumen. 
• This leaves the newly synthesized protein free in the lumen of the ER.

Figure 14-13.

Animation of co-translational insertion of protein into lumen of ER

Proteins inserted into membranes:

• Two categories of hydrophobic signals used in insertion of membrane proteins. All of these are membrane crossing domains:

  Start transfer sequences. These are of two types:

  • Signal peptide sequence - contains a cluster of about 8 -14 hydrophobic amino acids at the N-terminal end of a protein. This is the same as the signal peptide sequence mentioned above.
  • Start transfer sequence. Similar to a signal sequence, but located internally (not at the N terminal end of the protein). It also binds to the SRP and initiates transfer. Unlike the N-terminal signal sequence, it is not cleaved after transfer of the protein.
  • Stop transfer signal. This is also a sequence of about 8-14 hydrophobic amino acid residues. It follows either a N-terminal signal sequence or a start transfer sequence. 
    • The stop transfer signal is a membrane crossing domain. It remains in the membrane. 
    • When it is is encountered the translocation channel is disassembled. 
    • The peptide is not cleaved.
    • Translation continues in the cytoplasm.  
  Stop transfer signal. This is also a sequence of about 8-14 hydrophobic amino acid residues. 
  • It follows either a N-terminal signal sequence or a start transfer sequence. 
  • The stop transfer signal is a membrane crossing domain. It remains in the membrane. 
  • When it is is encountered the translocation channel is disassembled. 
  • The peptide is not cleaved.
  • Translation continues in the cytoplasm.  
• If a subsequent Start Transfer sequence is encountered in the protein, a second SRP binds to the start transfer sequence and a new translocation channel is opened in the membrane.

Figure 14-15.  Animation of insertion of a single pass membrane protein with N terminal end in ER lumen

Import of a membrane protein into the membrane of the ER. The blue sheath-like component shown in the figure is the translocation channel that moves the protein through the membrane. Notice that the Stop Transfer sequence (orange) results in the disassembly of the translocation channel. Note that the signal sequence at the N terminal end of the protein is cleaved off, releasing the N terminal portion of the protein into the ER lumen.  This example is a single pass membrane protein that contains a single membrane crossing domain (the stop transfer signal).

This diagram shows the relation between translocation control sequences (signal sequences, start transfer sequences, stop transfer sequences) and the arrangement of the protein in the membrane.  How would the translocation control sequences have to be arranged to get the N terminal end of  the protein on the cytoplasmic side? - to get the C terminal end of the protein on the cytoplasmic side?

Link to animations of co-translational insertion of proteins (5 different cases).