Biochemistry 3107 - Fall 1999

Protein Synthesis: Folding, Modification, Targeting and Degradation


Protein Folding

As they are being synthesized, proteins must adopt the correct conformation for their function.

Proteins may either fold spontaneously or they may need the assistance of chaperone proteins so that they do not get trapped in stable folding intermediates but rather fold into the correct final conformation.

There are 3 major classes of chaperones:


All are named because these proteins were first identified as Heat Shock Proteins - their synthesis increased in response to a sudden rise in temperature. In this role, chaperones protect important cell proteins from denaturation as a result of a sudden rise in temperature.


Post-Translational Modifications

Some proteins must be modified in one or more of a number of ways before they realize their final functional form. The following are some of the modifications that have been found to occur to proteins after they have have been synthesized:


Dealing with the N-terminal residue

In bacteria, the N-terminal residue of the newly-synthesized protein is modified in bacteria to remove the formyl group. The N-terminal methionine may also be removed.

In eukaryotes, the methionine is also subject to removal.


Amino Acid Modifications

Many of the amino-acid side-chains can be modified. Here are some examples:


The amino-terminal residues of some proteins are acetylated. For example, the N-terminal serine of histone H4 is invariably acetylated as are a number of lysine residues.



Phosphorylation of proteins (at Ser, Thr, Tyr and His residues) is an important regulatory mechanism. For example, the activity of glycogen phosphorylase is regulated by phosphorylation of Serine 14.

Phosphorylation of tyrosine residues is an important aspect of signal transduction pathways.

Bacterial cells sense and respond to environmental signals through histidine phosphorylation.



The activity of histones can be modified by methylation. Lysine 20 of histone H4 can be mono- or di- methylated.



The blood coagulation factor, prothrombin, contains a large number of carboxylated glutamatic acid residues in the N-terminal 32 amino acids. These modified residues are essential for activity. The modification requires vitamin K.



The conversion of proline to hydroxyproline in collagen is the classical example of a post-translational modification.

This conversion is catalyzed by prolyl-4-hydroxylase which is a tetramer of two a and two b subunits; the b subunits are multifunctional and also carry disulfide isomerase activity.

 Read more on the a OMIM entry [176710] and b OMIM entry [176790] subunits of prolyl-4-hydroxylase.




Many extracellular (but not intracellular) proteins are glycosylated. Mono- or Oligo-saccharides can be attached to asparagine (N-linked) or to serine/threonine (O-linked) residues.

Sugar residues are first assembled as a dolichol phosphate derivative. The sugars are then transferred to the appropriate site on a protein where some further modification may take place.

N-linked glycosylation takes place in the endoplasmic reticulum; O-linked glycosylation takes place in the Golgi apparatus.



Mononucleotide addition is used to regulate the activity of some enzymes. Two different examples are found among the system that regulates Nitrogen utilization in E. coli:

Glutamine synthetase is adenylylated (i.e. AMP is added) at a specific tyrosine residue. The enzyme is inactive when it is adenylylated. The degree of adenylylation is controlled by a regulatory protein, PII.

The ability of PII to regulate the adenylylation of glutamine synthetase is in turn regulated by its own uridylylation (i.e. the covalent addition of UMP). PII is also uridylylated at a tyrosine residue.


Lipid Addition

Some proteins have lipid moieties attached:

The viral src protein is myristoylated at the N-terminal glycine.

Rhodoposin is palmitoylated at a cysteine residue

The ras oncogene protein is farnesylated as are some G proteins.




The protein, thyroglobin, is iodinated during the synthesis of thyroxine.



Adding Prosthetic Groups

Proteins that require a prosthetic group for activity must have this group added. For example, the haem (heme) group must be added to globins and cytochromes; Fe-S clusters must be added to ferredoxins.


Forming Disulfide Bonds

Many extracellular proteins contain disulfide cross-links (intracellular proteins almost never do). The cross-links can only be established after the protein has folded up into the correct shape.

The formation of disulfide bonds is aided by the enzyme protein disulfide isomerase in eukaryotes and by the DsbA protein in bacteria. Protein disulfide isomerase is one of a number of activities found in the beta subunit (OMIM entry [176790] ) of the enzyme prolyl-4-hydroxylase.

Other activities of this subunit are:


Proteolytic Processing

Some proteins are synthesized as inactive precursor polypeptides which become activated only after proteolytic cleavage of the precursor polypeptide chain. Two well-known examples are:

Chymotrypsin & Trypsin

Chymotrypsin and trypsin are both synthesized as zymogens. Cleavage of chymotrypsinogen between Arg15 and Ile 16 by trypsin yields the enzymatically active pi-chymotrypsin. Two further proteolytic cleavages catalyzed by chymotrypsin removes the dipeptides Ser14-Arg15 and Thr147-Asn148 to yield alpha-chymotrypsin.

Trypsin is activated by the removal of the N-terminal seven amino acids.



Insulin is synthesized as a precursor polypeptide. The initial preproinsulin contains a signal sequence since the protein is targeted for secretion. The signal sequence is removed as described in the next section.

The resulting precursor, proinsulin, is converted into active insulin by specific peptidases which remove amino acids 31 - 63. The final form of the hormone has two polypeptide chains held by 2 interchain and 1 intrachain disulphide bonds.


Protein Targeting

Proteins that must be targetted within the cell must be intercepted early during synthesis so that this can happen correctly. As a protein is being synthesized, decisions must be taken about sending it to the correct location in the cell where it will be required. The information for doing this resides in the nascent protein sequence itself. Once the protein has reached its final destination, this information may be removed by proteolytic processing.

Targeting in Bacteria

In bacterial cells, the targeting decision is relatively straightforward: is the protein destined to be an intracellular protein or an extracellular one?

However, even here nuances are possible. In a gram-negative bacterium, such as E. coli, there must be some way of knowing whether a protein is destined to go to the cell membrane, the periplasmic space, or the outer membrane.

Secreted proteins contain a signal sequence. This is a short (6 - 30) stretch of hydrophobic amino acids, flanked on the N-terminal side by one or more positively charged amino acids such as lysine or arginine, and containing neutral amino acids with short side-chains (such as glycine or alanine) at the cleavage site. Some examples are:

E. coli Signal Sequences from the SwissProt Database
Entry Description Sequence


As proteins with signal sequences are synthesized, they are bound by the SecB protein. This prevents the protein from folding. SecB delivers the protein to the cell membrane where is is secreted through a pore formed by the SecE and SecY proteins. Secretion is driven by the SecA ATPase. After the protein has been secreted, the signal sequence is removed by a membrane bound leader peptidase.


Targeting in Eukaryotes

In eukaryotic cells, the situation is more complex. Extracellular proteins can be targeted for secretion, to the cell membrane, or to one of the many internal organelles. Intracellular proteins can be targeted for the cyoplasm, to the nucleus or to special organelles such as the mitochondrion or the chloroplast.

The Signal Sequence hypothesis was first enunciated by Gunther Blöbel who was awarded the Nobel Prize in Medicine in 1999 for his work.

The following diagram summarizes the choices/fates available to newly synthesized proteins in a eukaryotic cell:

Protein secretion in eukaryotic cells also involves a signal sequence. As in bacteria, the signal sequence is a short (6 - 30) stretch of hydrophobic amino acids, flanked on the N-terminal side by one or more positively charged amino acids such as lysine or arginine, and containing neutral amino acids with short side-chains (such as glycine or alanine) at the cleavage site:

Human Signal Sequences from the SwissProt Database
Entry # Description Sequence


Signal sequences are recognized by Signal Recognition Particles (SRPs) which are ribonucleoprotein particles containing a stable 305 nt 7S RNA and 6 polypeptides.

The 7S RNA has 2 domains - the Alu domain (so called because it is related to the Alu family of repeating sequences) and the S domain. The 7S RNA has 3 activities - a signal sequence recognition activity, an SRP receptor binding activity, and an elongation arrest activity.

As proteins are being synthesized, the N-terminal signal sequence is bound by the SRP particle. Protein synthesis is temporarily halted (elongation arrest) until the particle -- with the nascent polypeptide and the ribosome -- attaches to an SRP receptor complex in the Endoplasmic Reticulum membrane.

The SRP receptor is a heterodimer formed by a larger 68 kD alpha subunit and a smaller 30 kD subunit. The a subunit is a GTP-binding protein. Attachment of the ribosome triggers the release of GDP and binding of GTP. The GTP form of the SRP receptor binds the ribosome very tightly.

The ribosome is then transferred to a Ribosome receptor. Once again, the relatively slow hydrolysis of GTP allows time for this transfer to take place. Once transfer has taken place, GTP is hydrolyzed and the SRP receptor is free to interact with another SRP particle-ribosome complex.

Protein synthesis then resumes but now the nascent polypeptide is secreted into the lumen of the ER as it is being synthesized.

Shortly after the polypeptide enters the ER, signal peptidase cleaves the signal peptide. The finished polypeptide is then targeted through the ER and Golgi apparatus to the cell surface or to the lysosome.

Proteins that are synthesized on free ribosomes may also be targeted within the cell:

The following table summarizes some of the signals that target proteins within the eukaryotic cell:

Organelle Location of Signal in the Protein
Nature of Signal
Endoplasmic Reticulum C-terminus KDEL or HDEL (in yeast)
Mitochondrion N-terminal 3-5 nonconsecutive Arg or Lys; often contains Ser & Thr; never has acidic amino acids; no consensus sequence.
Chloroplast N-terminal Generally rich in Ser, Thr, and small hydrophobic amino acids; no acidic amino acids
Nucleus Internal Single cluster of 5 basic amino acids (e.g. KKKRK in the SV40 T antigen), or 2 smaller clusters separated by 10 amino acids
Lysosome Internal Covalent attachment of Mannose-6-phosphate
Peroxisome C-terminus SKF tripeptide at C-terminus


Protein Turnover

Protein lifetimes must also be regulated. Some proteins are needed for only very short times -- and could be harmful is present for too long. Others are needed all the time and it would be unnecessarily wasteful to keep re-synthesizing them.

The following table, taken from Table 30-10 of Biochemistry (2nd ed.) by Voet & Voet, demonstrates the great differences in lifetimes of some rat liver enzymes:

Enzyme Half-life (h)
Ornithine decarboxylase 0.2
RNA polymerase I 1.3
Tyrosine aminotransferase 2.0
Serine dehydratase 4.0
PEP carboxylase 5.0
Aldolase 118
cytochrome c 150


The lifetime of proteins in eukaryotes appears to be determined by the nature of the N-terminal amino acid. Some amino acids (e.g. Ala, Cys, Gly, Met, Pro, Ser, Thr, Val) stabilize proteins (at least in yeast); others (e.g. Arg, His, Ile, Leu, Lys, Phe, Trp, Tyr) destabilize proteins.

In bacteria, the C-terminal amino acid also has an effect on the half-life of a protein.

Protein turnover in the eukaryotic cell cytoplasm involves the very highly conserved protein ubiquitin. This small (76 aa) protein is covalently attached to any protein selected for degradation. Such proteins are then degraded by the 26S proteosome.


Format and Original Material © Martin E. Mulligan, 1996-99