The Endomembrane System

Overview: The endomembrane system consists of the Endoplasmic Reticulum, the Golgi apparatus, Lysosomes, Endosomes and Secretory Vesicles. These compartments are involved in the processing of proteins for export from the cell, proteins destined for lysosomes, and proteins entering the cell from the cell surface. Once proteins enter the endoplasmic reticulum they never return to the cytosol compartment; they are carried by vesicle transport to the other compartments of the system. This flow of vesicles is highly regulated. Overview of the endomembrane system outlining the three major parts of the system. There are three major subdivisions of the endomambrane system the secretory pathway the lysosomal pathway and the endocytotic pathway Proteins entering the secretory or lysosomal are synthesized on ribosomes in the cytosol and are then transferred to the endoplasmic teticulum

Protein targeting to the Endoplasmic Reticulum

1. Proteins entering the ER

Overview: Synthesis of all proteins begins in the cytosol compartment. For proteins entering the secretory or Lysosomal pathways, the first step is targeting to the endoplasmic reticulum. This targeting relies on a targeting signal encoded in the N terminal portion of the protein. The targeting signal is recognized by a specific receptor that results in the protein entering the endoplasmic reticulum.

1. Targeting of Proteins 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 amino 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 reticuluum.

Proteins secretory or lysosomal pathways enter the ER and don't come out again. The proteins entering either of these pathways may be of either of two types:

• Proteins that are completely translocated into the endoplasmic reticuluum. These proteins are soluble (not membrane proteins) and are destined for secretion, or for transfer to lysosomes. In all of these cases the 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, membranes of another organelle (Golgi, lysosomes or endosomes), or the plasma membrane. In all of these cases the proteins stay within the membrane once they are inserted into the ER membrane (e.g. cellulose synthase).

Translation of all proteins begins on free ribosomes. Those ribosomes that produce proteins for export through the endoplasmic reticulum become attached to the endoplasmic reticulum as ribosomes of the rough ER. The signal for ER entry is 8 or more hydrophobic amino acid residues (Table 14-3) which rivets the polypeptide to the ER membrane and is also involved in translocation.

Whether or not a ribosome becomes attached to the endoplasmic reticulum depends on the nature of the message being translated, the protein being made, and is not an intrinsic property of the ribosome itself. The ribosome and its attached nascent peptide become targeted to the endoplasmic reticulum.

  Figure 14-12

Targeting to the endoplasmic reticulum takes place through the interaction of the signal peptide sequence ( a sequence of at least eight hydrophobic amino acids at the amino terminal end of the polypeptide. 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.

Figure 14-13. shows two components.

1. There is a Signal Recognition Particle (SRP) in the cytosol. This binds to the ER Signal sequence when it is exposed on the ribosome and slows protein synthesis long enough to allow the SRP to find the second part, the SRP Receptor.

2. The Signal Recognition Particle Receptor (SRPR) which is embedded in the ER membrane. We now have the new polypeptide synthesizing system in place and protein synthesis speeds up. It seems that the Signal Sequence opens the translocation channel.

Experimental test that ER targeting signal is both necessary and sufficient to bring about targeting.

Figure 14-6 experimental test of the role of signal sequences. IMPORTANT

How do proteins get into the ER?

SOLUBLE PROTEINS

The peptide moves through the translocation channel into the lumen of the ER. The signal peptide sequence remains attached to the membrane. It is later cleaved off by a signal peptidase. Leaving the protein free in the lumen of the ER.

Figure 14-14

Animation of this process

MEMBRANE PROTEINS

Key point is that the orientation of a protein in the membrane is established  when it is first inserted into the membrane. This orientation of the protein persists all of the way to its final destination. That is, the cytosolic side of membrane remains on the cytosolic side throughout all processes.

As membrane proteins are being translated, they are translocated  or transferred into the ER until a hydrophobic membrane crossing domain is encountered. This serves as a 'stop transfer' signal and leaves the protein inserted in the ER membrane.

Figure 14-15. Animation of this process

Import of a membrane protein. This figure illustrates the case of a protein being incorporated in the membrane of the endoplasmic reticulum, but import into organellar membranes works much the same way. The blue sheath-like component shown in the figure is the transport complex that moves the protein through the membrane. This example is a single pass membrane protein that contains a single membrane crossing domain.

The hydrophobic trans-membrane domain holds the protein in the membrane because of the very strong hydrophobic interaction between this part of the protein and the hydrophobic membrane core.

Try this excellent link to web site dealing with insertion of proteins in membrane.

Proteins with multiple membrane crossing domains are inserted in the the membrane through the action of multiple pairs of start transfer and stop transfer signals:

Click to enlarge Animation of this process

Fig. 14-16 insertion of a double pass membrane protein into the membrane. The signal sequence is not at the N terminus and is not removed. Transfer continues until a stop signal is reached.   There may be more than one pair of start and stop transfer signals. Transfer is reinitiated with each start transfer signal. This means that at each transfer stop signal (membrane crossing domain) the ribosome becomes detached from the ER membrane. If later a start transfer sequence is encounters, it binds to a new SRP and forms a new association between the ribosome and the ER membrane that leads to the insertion of the start transfer sequence and the following amino acids up to and including either the C terminal end or a stop transfer sequence, which ever is encountered first.

There are two major 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:
  • N-terminal signal peptide sequence - a cluster of about 8 hydrophobic amino acids at the N-terminal end of a protein. This sequence remains in the membrane and is cleaved off of the  protein after transfer through the membrane.
  • Internal 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 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. The peptide is not cleaved.

This process of membrane insertion has a very important result: It establishes orientation of membrane proteins. Recall the earlier discussion of 'sidedness of membranes'. This is one of the chief ways that 'sidedness' happens.

Notice that the C-terminal end of the protein is on the cytosolic side of the membrane and the N-terminal end is not in the cytosol, but on the inside of the ER, or organelle.

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?

Now look at what happens if the protein is incorporated into a vesicle and later fused with the plasma membrane: The cytosolic side remains in the cytosol. This is a key idea.

Insertion of membrane proteins and sidedness of membranes. In A, a protein is inserted into the ER membrane. The cytosolic side of the membrane is labled 'C'. If a vesicle is budded off of the ER containing the protein, the cytosolic portion is still in the cytosol (Figure B). If the vesicle fuses with the plasma membrane (Figure C) the same relationships are maintained, resulting in the incorporation of the protein into the plasma membrane (Figure D) with the same orientation (C vs I) that it had when it first entered the membrane.

Can you do this one? Make a map of each of the proteins shown at A and B in the figure at the left. Indicate N terminal end, the relative location of any signal sequences. start transfer sequences, stop transfer sequences, and the C terminal end. The membrane crossing domains are shown in red. Explain the sequence of events leading to insertion of each of these proteins in the membrane (This is a very important test of understanding) See Fig. 14-15 and 14-16 for hints.