Endoplasmic reticulum (ER), together with the Golgi apparatus, endosomes, lysosomes and transport vesicles, comprise the endomembrane system for protein trafficking. If a protein is required to be secreted, or localised at any part of the secretory pathway, its trafficking begins at the ER. It is important to note that ER is not only the entry site for protein secretion, but is also a major site of protein modification and folding. 


Protein entry into the ER

ER coated with synthesising ribosomes is called rough endoplasmic reticulum, however rough and smooth ER are not considered to be different structures. Usually, ER has smooth and rough regions, depending on cell type and condition.

Proteins pass across the ER membrane by a process called co-translational translocation. In other words, as a protein is synthesised by ribosomes it gets threaded across the membrane. 

Ribosomes are targeted to ER cytoplasmic face by signal recognition particle (SRP) machinery. Each protein destined to cross the membrane has a start translocation sequence - somewhat similar to a local postcode. The postcode to enter the ER is a stretch of approximately 8 hydrophobic amino acids. As the ribosomes begin peptide translation in the cytosol, newly synthesised N-terminus end of the peptide - with the ER postcode - is bound by the SRP. SRP then binds SRP-receptor in the ER membrane, anchoring the ribosome to ER. Next, SRP-receptor targets the synthesising ribosome to a translocon; a multiprotein complex that spans the ER membrane. Sec61, a pore-forming part of the translocon is opened by the N-terminal postcode and the protein gets threaded across the membrane.


If the synthesised protein is soluble, the signal sequence gets cleaved after translocation by a signal peptidase and the protein diffuses into the ER's lumen. 


Cleavage by signal peptidase

Alternatively, a protein may be destined to remain in the ER membrane.

There are several different ways that the protein can be inserted into the lipid bilayer. Stop sequences anchor the peptide to the membrane when the signal sequence gets cleaved.

A peptide may possess a single stop sequence, or a start sequence may also act as a stop sequence.

Finally, a peptide may possess multiple start and stop sequences, which will result in multiple transmembrane domains. The number of stop and start sequences will dictate how many times the peptide binds to the membrane.

Three modes of peptide insertion into the membrane are illustrated below.


Three modes of membrane integration

Post-translational modifications

Many proteins, while being translocated, are converted to glycoproteins by the addition of short oligosaccharide side chains to the N-terminus. 


N-terminal glycosylation is catalysed by a membrane-bound enzyme complex – oligosaccharyl transferase.  This enzyme recognises the Asn-X-Ser/Thr motif, and covalently attaches an oligosaccharide to the asparagine side chain. Dolichol is another membrane-bound protein, which serves as an oligosaccharide carrier and donor for N-linked glycosylation.


N-linked glycosylation

Another common post-translational modification is disulphide bond formation by protein disulphide isomerase (PDI). The enzyme recognises two cysteine sulfhydryl groups (-SH) and oxidises them to form either intramolecular (between the residues of a same peptide) or intermolecular (between two peptides) disulphide bonds (S-S).



Molecular chaperones


PDI can also reduce existing mispaired disulphide bonds and therefore works as a molecular chaperone.

There are numerous molecular chaperones in the ER lumen, which assist in protein folding, prevent aggregation and reduce unfolded protein stress. Chaperones bind to newly-made peptides and dissociate once they are correctly folded. Removal of misfolded peptides is also a form of ‘quality control'. Misfolded proteins can be dangerous for the cell and thus are retained in ER lumen by the chaperones.  One such example is the binding immunoglobulin protein(BiP). BiP recognises hydrophobic parts of the newly synthesised protein and uses ATP to bind them and prevent peptide aggregation.

Not all chaperones use ATP to assist in protein folding. Calnexin and calreticulin are members of lectin (sugar-binding) chaperones that bind to and prevent the export of misfolded N-glycosylated proteins. The process is mediated by glucose. Misfolded proteins have glucose attached to their oligosaccharyl chain that lectins recognise and direct the peptide for glucose removal and another round of folding. The glucose molecule therefore serves as a tag for misfolded proteins. Once the protein is correctly folded, it loses the glucose and is no longer bound by the lectins.


Chaperone-assisted folding:


Chaperone-assisted folding

Molecular chaperones

Misfolded proteins


Despite the presence of chaperones in the ER lumen, many peptides still fail to fold due to transcription or translation errors. To prevent a protein plaque building up in the ER, these proteins are exported back to the cytosol and degraded. The process is called ER assisted degradation (ERAD). When a mutated protein fails to fold and is retained by lectins many times, it gets translocated back to the cytosol where it is tagged with ubiquitin. This signals that the protein must be degraded by a proteasome. 

Cells carefully monitor the amount of unfolded proteins in ER, and any increase will trigger an unfolded protein response (UPR). When the number of unfolded peptides builds up, three parallel UPR pathways are activated depending on the cell type.

The first pathway involves transmembrane receptor kinase activation, which in turn up-regulates chaperones.

The second pathway involves activating a different transmembrane kinase, which results in reduced translation and hence reduced flux of proteins into the ER.

Finally, the third UPR pathway involves the release of ER-resident transcription factors of UPR proteins.

The ultimate result, regardless of which pathway is activated, is reduced ER stress. If, however, the stress is still too heavy, cells often commit apoptosis (cell 'suicide').



  • Protein trafficking, unless it is a cytosolic protein, starts at the ER
  • Proteins are directed to ER by an 8 hydrophobic amino acid sequence, which is recognised by SRP machinery
  • ER is a major site for protein folding and modification
  • ER chaperones assist in folding and retain misfolded proteins
  • ER stress triggers unfolded protein response
  • Too much ER stress can lead to apoptosis


Further reading

BRAAKMAN, I. & BULLEID, N. J. 2011. Protein Folding and Modification in the Mammalian Endoplasmic Reticulum. In: KORNBERG, R. D., RAETZ, C. R. H., ROTHMAN, J. E. & THORNER, J. W. (eds.) Annual Review of Biochemistry, Vol 80. Palo Alto: Annual Reviews.

LUIRINK, J. & SINNING, I. 2004. SRP-mediated protein targeting: structure and function revisited. Biochimica Et Biophysica Acta-Molecular Cell Research, 1694, 17-35.

SCHRODER, M. & KAUFMAN, R. J. 2005. ER stress and the unfolded protein response. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 569, 29-63.



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