Translation

A step in protein biosynthesis wherein the genetic code carried by mRNA is

decoded

to

produce

the

specific

sequence

of amino

acids in

a polypeptide chain.

Prokaryotic translation
Prokaryotic

translation is

the

process

by

which messenger

RNA is translated into proteins in prokaryotes.

Initiation
Initiation of translation in prokaryotes involves the assembly of the
components of the translation system, which are: the two ribosomal subunits
(50S and 30S subunits); the mature mRNA to be translated; the tRNA
charged with N-formylmethionine (the first amino acid in the nascent
peptide); guanosine
prokaryotic

triphosphate (GTP)

elongation

factor EF-P and

as

source

the

three

of

energy;

prokaryotic

the

initiation

factors IF1, IF2, and IF3, which help the assembly of the initiation complex.
Variations in the mechanism can be anticipated.
The ribosome has three active sites: the A site, the P site, and the E site.
The A site is the point of entry for the aminoacyl tRNA (except for the first
aminoacyl tRNA, which enters at the P site). The P site is where the peptidyl
tRNA is formed in the ribosome. And the E site which is the exit site of the
now uncharged tRNA after it gives its amino acid to the growing peptide
chain.
The selection of an initiation site (usually an AUG codon) depends on the
interaction between the 30S subunit and the mRNA template. The 30S
subunit binds to the mRNA template at a purine-rich region (the ShineDalgarno sequence) upstream of the AUG initiation codon. The ShineDalgarno sequence is complementary to a pyrimidine rich region on the 16S
rRNA component of the 30S subunit. This sequence has been evolutionarily

conserved and plays a major role in the mirobial world we know today.
During the formation of the initiation complex, these complementary
nucleotide sequences pair to form a double stranded RNA structure that
binds the mRNA to the ribosome in such a way that the initiation codon is
placed at the P site.
Elongation
Elongation of the polypeptide chain involves addition of amino acids to
the carboxyl end

of

the

growing

chain.

The

growing protein exits

the ribosome through the polypeptide exit tunnel in the large subunit.[1]
Elongation

starts

when

the

fMet-tRNA

enters

the

site,

causing

a conformational change which opens the A site for the new aminoacyl-tRNA
to

bind.

This

binding

is

facilitated

byelongation

factor-Tu (EF-Tu),

small GTPase. For fast and accurate recognition of the appropriate tRNA, the
ribosome

utilizes

large

conformational

changes

(conformational

proofreading) .[2] Now the P site contains the beginning of the peptide chain
of the protein to be encoded and the A site has the next amino acid to be
added to the peptide chain. The growing polypeptide connected to the tRNA
in the P site is detached from the tRNA in the P site and a peptide bond is
formed between the last amino acids of the polypeptide and the amino acid
still attached to the tRNA in the A site. This process, known as peptide bond
formation, is catalyzed by a ribozyme (the 23S ribosomal RNA in the 50S
ribosomal subunit). Now, the A site has the newly formed peptide, while the
P site has an uncharged tRNA (tRNA with no amino acids). The newly formed
peptide in the A site tRNA is known as dipeptide and the whole assembly is
called dipeptidyl-tRNA. The tRNA in the P site minus the amino acid is known
to be deacylated. In the final stage of elongation, called translocation,
the deacylated tRNA (in the P site) and the dipeptidyl-tRNA (in the A site)
along with its corresponding codons move to the E and P sites, respectively,
and a new codon moves into the A site. This process is catalyzed
by elongation factor G (EF-G). The deacylated tRNA at the E site is released

from the ribosome during the next A-site occupation by an aminoacyl-tRNA

again facilitated by EF-Tu.[3]
The ribosome continues to translate the remaining codons on the mRNA as
more aminoacyl-tRNA bind to the A site, until the ribosome reaches a stop
codon on mRNA(UAA, UGA, or UAG).
The translation machinery works relatively slowly compared to the enzyme
systems

that

catalyze

DNA

replication.

Proteins

in

prokaryotes

are

synthesized at a rate of only 18 amino acid residues per second, whereas

bacterial replisomes synthesize DNA at a rate of 1000 nucleotides per
second. This difference in rate reflects, in part, the difference between
polymerizing

four

types

of

nucleotides

to

make

nucleic

acids

and

polymerizing 20 types of amino acids to make proteins. Testing and rejecting

incorrect aminoacyl-tRNA molecules takes time and slows protein synthesis.
In bacteria, translation initiation occurs as soon as the 5' end of an mRNA is
synthesized, and translation and transcription are coupled. This is not
possible in eukaryotes because transcription and translation are carried out
in separate compartments of the cell (the nucleus and cytoplasm).
Termination
Termination occurs when one of the three termination codons moves into the
A site. These codons are not recognized by any tRNAs. Instead, they are
recognized by proteins called release factors, namely RF1 (recognizing the
UAA and UAG stop codons) or RF2 (recognizing the UAA and UGA stop
codons). These factors trigger the hydrolysis of theester bond in peptidyltRNA and the release of the newly synthesized protein from the ribosome. A
third release factor RF-3 catalyzes the release of RF-1 and RF-2 at the end of
the termination process.
Recycling
The post-termination complex formed by the end of the termination step
consists of mRNA with the termination codon at the A-site, an uncharged

tRNA in the P site, and the intact 70S ribosome. Ribosome recycling step is
responsible for the disassembly of the post-termination ribosomal complex.
[4]

Once the nascent protein is released in termination,Ribosome Recycling

Factor and Elongation Factor G (EF-G) function to release mRNA and tRNAs
from ribosomes and dissociate the 70S ribosome into the 30S and 50S
subunits. IF3 then replaces the deacylated tRNA releasing the mRNA. All
translational components are now free for additional rounds of translation.

Polysomes
Translation is carried out by more than one ribosome simultaneously.
Because of the relatively large size of ribosomes, they can only attach to
sites on mRNA 35 nucleotides apart. The complex of one mRNA and a
number of ribosomes is called a polysome or polyribosome.[citation needed]
Regulation of Translation
When bacterial cells run out of nutrients, they enter stationary phase and
downregulate protein synthesis. Several processes mediate this transition.
[5]

For instance, in E. coli, 70S ribosomes form 90S dimers upon binding with

small

6.5

kDa

protein, ribosome

modulation

factor RMF.[6][7] These

intermediate ribosome dimers can subsequently bind ahibernation promotion

factor (the 10.8 kDa protein, HPF) molecule to form a mature 100S ribosomal
particle, in which the dimerization interface is made by the two 30S subunits
of the two participating ribosomes.[8] The ribosome dimers represent a
hibernation state and are translationally inactive. [9] A third protein that can
bind

to

ribosomes

when E.

coli cells

enter

the

stationary

phase

is YfiA (previously known as RaiA).[10] HPF and YfiA are structurally similar,
and both proteins can bind to the catalytic A- and P-sites of the ribosome. [11]
[12]

RMF blocks ribosome binding to mRNA by preventing interaction of the

messenger with 16S rRNA.[13] When bound to the ribosomes the C-terminal
tail ofE. coli YfiA interferes with the binding of RMF, thus preventing

dimerization and resulting in the formation of translationally inactive

monomeric 70S ribosomes.[13][14]

Mechanism of ribosomal subunit dissociation by RsfS (= RsfA)

In addition to ribosome dimerization, the joining of the two ribosomal
subunits can be blocked by RsfS (formerly called RsfA or YbeB). [15]RsfS binds
to L14, a protein of the large ribosomal subunit, and thereby blocks joining of
the small subunit to form a functional 70S ribosome, slowing down or
blocking translation entirely. RsfS proteins are found in almost all eubacteria
(but

not archaea)

and

homologs

are

present

in mitochondria and chloroplasts (where they are called C7orf30 and iojap,
respectively). However, it is not known yet how the expression or activity of
RsfS is regulated.
Another ribosome-dissociation factor in Escherichia coli is HflX, previously a
GTPase of unknown function. Zhang et al. (2015) showed that HflX is a heat
shockinduced ribosome-splitting factor capable of dissociating vacant as
well as mRNA-associated ribosomes. The N-terminal effector domain of HflX
binds to the peptidyl transferase center in a strikingly similar manner as that
of the class I release factors and induces dramatic conformational changes in
central

intersubunit

bridges,

thus

promoting

subunit

dissociation.

Accordingly, loss of HflX results in an increase in stalled ribosomes upon heat

shock and possiblu other stress conditions.[16]

Eukaryotic Translation

The broad outlines of eukaryotic protein synthesis are the same as in

prokaryotic protein synthesis. The genetic code is generally the same (some
microorganisms and eukaryotic mitochondria use slightly different codons),
rRNA and protein sequences are recognizably similar, and the same set of
amino acids is used in all organisms. However, specific differences exist
between the two types of protein synthesis at all steps of the process.
Initiation
Both prokaryotes and eukaryotes initiate protein synthesis with a specialized
methionyltRNA in response to an AUG initiation codon. Eukaryotes, however,
use an initiator mettRNA

met

that is not formylated. Recognition of the

initiator AUG is also different. Only one coding sequence exists per
eukaryotic mRNA, and eukaryotic mRNAs are capped. Initiation, therefore,
uses a specialized cap binding initiation factor to position the mRNA on the
small ribosomal subunit. Usually, the first AUG after the cap (that is, 3 to it)
is used for initiation.
Elongation
Most differences in elongation result from the fact that the eukaryotic cell
has different compartments, which are separated by membranes. Both
prokaryotic and eukaryotic cells, of course, have an inside and outside;
however, eukaryotic proteins can be targeted to, for example, the
mitochondrion.
Translating ribosomes in eukaryotes are located in different places in the cell
depending on the fate of their proteins. Free polysomes are in the cytoplasm
and synthesize cytoplasmic proteins and those that are bound for most
intracellular organelles, for example, the nucleus. Members of the second
class of polysomes, membranebound polysomes, are attached to the
endoplasmic reticulum (forming the rough ER), and synthesize exported
proteins. In cells that are actively secreting enzymes or hormones (for
example, those in the pancreas), most of the protein synthesis occurs on the
rough ER.

The messages encoding exported proteins must be recognized. For example,

the digestive proteases are made in the pancreas. If they are released into
the pancreas rather than the intestine, they will selfdigest the organ that
makes them. Carrying out this export efficiently is obviously important.
The signal

hypothesis explains

how

proteins

destined

for

export

are

discriminated. Proteins that are destined for export contain a short (less than
30 amino acids long) sequence made up of hydrophobic amino acids at their
amino terminus. Because peptide synthesis occurs in the aminotocarboxy
direction, the signal peptide is the first part of the protein that is made.
Signal peptides are not found in most mature secreted proteins because they
are cleaved from the immature proteins during the secretion and maturation
process.

See

Figure 1.

The

process

of

protein

export

involves

small,

cytoplasmic

ribonucleoprotein particle (the Signal Recognition Particle or SRP) with the

signal coding mRNA sequence and/or the signal peptide itself. This
interaction stops translation of the protein. Then, the stalled or arrested
ribosome moves to the endoplasmic reticulum (ER). A receptor on the ER
binds the SRP.
Once the complex containing ribosome, mRNA, signal peptide, and SRP is
docked onto the membrane, SRP leaves the complex and the ribosome
resumes translation. The signal peptide inserts across the membrane; this
insertion is dependent on the hydrophobic nature of the signal sequence.
The rest of the protein follows the signal sequence across the membrane, like
thread through the eye of a needle. The protein folds into its secondary and
tertiary structure in the lumen(inside cavity) of the ER. The signal sequence
is cut away from the protein either during translation (cotranslational
processing) or, less often, after the protein is released from the ribosome
(posttranslational processing). After the polypeptide chain is completed, the
ribosome is released from the ER and is ready to initiate synthesis of a new
protein. Secreted proteins are also made by prokaryotes by using a signal
sequence mechanism, with the cell membrane taking the place of the ER
membrane.
This scheme can also accommodate the synthesis of membranebound
proteins. In this case, the protein is not released into the lumen of the ER,
but rather stays bound to the membrane. One or more anchor sequences (or
stoptransfer sequences) in the newly made protein keeps the partially
folded region of the protein in the membrane.
Protein glycosylation
Many eukaryotic membranebound and secreted proteins contain a complex
of sugar residues bound to the side chains of either asparagine to make N
linked sugar residues or serine and threonine to make Olinked sugar

residues. The core of the glycosyl complex is assembled, not by adding

sugars one after another to the protein chain, but by synthesizing the core
oligosaccharide on a membrane lipid, dolichol phosphate.

Dolichol is a long chain of up to twenty 5carbon isoprenoid units. The core

oligosaccharide assembled on dolicol phosphate ultimately contains 14
saccharides that are transferred all at once to an asparagine residue on the
protein. After the glycoprotein is assembled, the core oligosaccharide is
trimmed by removal of the three glucose residues from the end, making
a highmannose oligosaccharide

on

the

protein.

The

highmannose

oligosaccharide is moved from the ER for further modification in the Golgi

complex, a structure composed of layered intracellular membranes. The
completed glycoprotein moves to the plasma membrane in a membrane
granule, which buds off from the Golgi. The granule fuses with the plasma
membrane to release its contents. Binding to receptors in the plasma
membrane brings some lysosomal enzymes back into the cell. The enzyme
receptor complex is taken into the cell in a process that is essentially the
reverse of secretion, although it involves different membrane proteins.

Overview of Post-Translational Modifi cations (PTMs)

o

Pierce Protein Methods

Protein post-translational modification (PTM) increases the functional

diversity of the proteome by the covalent addition of functional groups
or proteins, proteolytic cleavage of regulatory subunits or degradation
of

entire

proteins.

glycosylation,

These

ubiquitination,

modifications
nitrosylation,

include

phosphorylation,

methylation,

acetylation,

lipidation and proteolysis and influence almost all aspects of normal cell
biology and pathogenesis. Therefore, identifying and understanding
PTMs is critical in the study of cell biology and disease treatment and
prevention.
Introduction
Within the last few decades, scientists have discovered that the human
proteome is vastly more complex than the human genome. While it is
estimated that the human genome comprises between 20,000 and 25,000
genes (1), the total number of proteins in the human proteome is estimated
at over 1 million (2). These estimations demonstrate that single genes
encode multiple proteins. Genomic recombination, transcription initiation at
alternative promoters, differential transcription termination, and alternative
splicing of the transcript are mechanisms that generate different mRNA
transcripts from a single gene (3).
The increase in complexity from the level of the genome to the proteome is
further facilitated by protein post-translational modifications (PTMs). PTMs
are chemical modifications that play a key role in functional proteomics,
because they regulate activity, localization and interaction with other cellular
molecules such as proteins, nucleic acids, lipids, and cofactors.
Post-translational modifications are key mechanisms to increase proteomic
diversity. While the genome comprises 20-25,000 genes, the proteome is
estimated

to

encompass

over

million

proteins.

Changes

at

the

transcriptional and mRNA levels increase the size of the transcriptome

relative to the genome, and the myriad of different post-translational

modifications exponentially increases the complexity of the proteome
relative to both the transcriptome and genome.
Additionally, the human proteome is dynamic and changes in response to a
legion

of

stimuli,

and

post-translational

modifications

are

commonly

employed to regulate cellular activity. PTMs occur at distinct amino acid side
chains or peptide linkages and are most often mediated by enzymatic
activity. Indeed, it is estimated that 5% of the proteome comprises enzymes
that perform more than 200 types of post-translational modifications (4).
These enzymes include kinases, phosphatases, transferases and ligases,
which add or remove functional groups, proteins, lipids or sugars to or from
amino acid side chains, and proteases, which cleave peptide bonds to
remove specific sequences or regulatory subunits. Many proteins can also
modify themselves using autocatalytic domains, such as autokinase and
autoprotolytic domains.
Post-translational modification can occur at any step in the "life cycle" of a
protein. For example, many proteins are modified shortly after translation is
completed to mediate proper protein folding or stability or to direct the
nascent protein to distinct cellular compartments (e.g., nucleus, membrane).
Other modifications occur after folding and localization are completed to
activate or inactivate catalytic activity or to otherwise influence the
biological activity of the protein. Proteins are also covalently linked to tags
that target a protein for degradation. Besides single modifications, proteins
are often modified through a combination of post-translational cleavage and
the addition of functional groups through a step-wise mechanism of protein
maturation or activation.
Protein PTMs can also be reversible depending on the nature of the
modification. For example, kinases phosphorylate proteins at specific amino
acid side chains, which is a common method of catalytic activation or

inactivation. Conversely, phosphatases hydrolyze the phosphate group to

remove it from the protein and reverse the biological activity. Proteolytic
cleavage of peptide bonds is a thermodynamically favorable reaction and
therefore permanently removes peptide sequences or regulatory domains.
Consequently,

the

analysis

of

proteins

and

their

post-translational

modifications is particularly important for the study of heart disease, cancer,

neurodegenerative diseases and diabetes. The characterization of PTMs,
although challenging, provides invaluable insight into the cellular functions
underlying etiological processes. Technically, the main challenges in studying
post-translationally modified proteins are the development of specific
detection and purification methods. Fortunately, these technical obstacles
are being overcome with a variety of new and refined proteomics
technologies.

Post-Translational Modifications
As noted above, the large number of different PTMs precludes a thorough
review of all possible protein modifications. Therefore, this overview only
touches on a small number of the most common types of PTMs studied in
protein

research

today.

Furthermore,

greater

focus

is

placed

on

phosphorylation, glycosylation and ubiquitination, and therefore these PTMs

are described in greater detail on pages dedicated to the respective PTM.
Phosphorylation
Reversible protein phosphorylation, principally on serine, threonine or
tyrosine residues, is one of the most important and well-studied posttranslational modifications. Phosphorylation plays critical roles in the
regulation of many cellular processes including cell cycle, growth, apoptosis
and signal transduction pathways.

Glycosylation
Protein glycosylation is acknowledged as one of the major post-translational
modifications, with significant effects on protein folding, conformation,
distribution, stability and activity. Glycosylation encompasses a diverse
selection of sugar-moiety additions to proteins that ranges from simple
monosaccharide modifications of nuclear transcription factors to highly
complex branched polysaccharide changes of cell surface receptors.
Carbohydrates in the form of aspargine-linked (N-linked) or serine/threoninelinked (O-linked) oligosaccharides are major structural components of many
cell surface and secreted proteins.
Ubiquitination
Ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids that is
appended to the -NH2 of lysine in target proteins via the C-terminal glycine
of ubiquitin. Following an initial monoubiquitination event, the formation of a
ubiquitin polymer may occur, and polyubiquitinated proteins are then
recognized by the 26S proteasome that catalyzes the degradation of the
ubiquitinated protein and the recycling of ubiquitin.
S-Nitrosylation
Nitric oxide (NO) is produced by three isoforms of nitric oxide synthase (NOS)
and is a chemical messenger that reacts with free cysteine residues to form
S-nitrothiols (SNOs). S-nitrosylation is a critical PTM used by cells to stabilize
proteins, regulate gene expression and provide NO donors, and the
generation, localization, activation and catabolism of SNOs are tightly
regulated.
S-nitrosylation is a reversible reaction, and SNOs have a short half life in the
cytoplasm because of the host of reducing enzymes, including glutathione
(GSH) and thioredoxin, that denitrosylate proteins. Therefore, SNOs are often

stored in membranes, vesicles, the interstitial space and lipophilic protein

folds to protect them from denitrosylation (5). For example, caspases, which
mediate apoptosis, are stored in the mitochondrial intermembrane space as
SNOs. In response to extra- or intracellular cues, the caspases are released
into

the

cytoplasm,

and

the

highly

reducing

environment

rapidly

denitrosylates the proteins, resulting in caspase activation and the induction

of apoptosis.
S-nitrosylation is not a random event, and only specific cysteine residues are
S-nitrosylated. Because proteins may contain multiple cysteines and due to
the labile nature of SNOs, S-nitrosylated cysteines can be difficult to detect
and distinguish from non-S-nitrosylated amino acids. The biotin switch assay,
developed by Jaffrey et al., is a common method of detecting SNOs, and the
steps of the assay are listed below (6):

All free cysteines are blocked.

All remaining cysteines (presumably only those that are denitrosylated)

are denitrosylated.

The now-free thiol groups are then biotinylated.

Biotinylated proteins are detected by SDS-PAGE and Western blot

analysis or mass spectrometry (7).

Methylation

The transfer of one-carbon methyl groups to nitrogen or oxygen (N- and Omethylation,

respectively)

to

amino

acid

side

chains

increases

the

hydrophobicity of the protein and can neutralize a negative amino acid

charge when bound to carboxylic acids. Methylation is mediated by
methyltransferases, and S-adenosyl methionine (SAM) is the primary methyl
group donor.

Methylation occurs so often that SAM has been suggested to be the mostused substrate in enzymatic reactions after ATP (4). Additionally, while Nmethylation

is

irreversible,

O-methylation

is

potentially

reversible.

Methylation is a well-known mechanism of epigenetic regulation, as histone

methylation and demethylation influences the availability of DNA for
transcription. Amino acid residues can be conjugated to a single methyl
group or multiple methyl groups to increase the effects of modification.
N-Acetylation
N-acetylation, or the transfer of an acetyl group to nitrogen, occurs in almost
all eukaryotic proteins through both irreversible and reversible mechanisms.
N-terminal acetylation requires the cleavage of the N-terminal methionine by
methionine aminopeptidase (MAP) before replacing the amino acid with an
acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes. This
type of acetylation is co-translational, in that N-terminus is acetylated on
growing polypeptide chains that are still attached to the ribosome. While 8090% of eukaryotic proteins are acetylated in this manner, the exact
biological significance is still unclear (4).
Acetylation at the -NH2 of lysine (termed lysine acetylation) on histone Ntermini is a common method of regulating gene transcription. Histone
acetylation is a reversible event that reduces chromosomal condensation to
promote transcription, and the acetylation of these lysine residues is
regulated by transcription factors that contain histone acetyletransferase
(HAT)

activity.

While

transcription

factors

with

HAT

activity

act

as

transcription co-activators, histone deacetylase (HDAC) enzymes are corepressors that reverse the effects of acetylation by reducing the level of
lysine acetylation and increasing chromosomal condensation.
Sirtuins (silent information regulator) are a group of NAD-dependent
deacetylases that target histones. As their name implies, they maintain gene

silencing by hypoacetylating histones and have been reported to aid in

maintaining genomic stability (8).
While acetylation was first detected in histones, cytoplasmic proteins have
been reported to also be acetylated, and therefore acetylation seems to play
a greater role in cell biology than simply transcriptional regulation (9).
Furthermore, crosstalk between acetylation and other post-translational
modifications, including phosphorylation, ubiquitination and methylation, can
modify the biological function of the acetylated protein (10).
Protein acetylation can be detected by chromosome immunoprecipitation
(ChIP) using acetyllysine-specific antibodies or by mass spectrometry, where
an increase in histone by 42 mass units represents a single acetylation.
Lipidation
Lipidation is a method to target proteins to membranes in organelles
(endoplasmic reticulum [ER], Golgi apparatus, mitochondria), vesicles
(endosomes, lysosomes) and the plasma membrane. The four types of
lipidation are:

C-terminal glycosyl phosphatidylinositol (GPI) anchor

N-terminal myristoylation

S-myristoylation

S-prenylation

Each type of modification gives proteins distinct membrane affinities,

although all types of lipidation increase the hydrophobicity of a protein and
thus its affinity for membranes. The different types of lipidation are also not

mutually exclusive, in that two or more lipids can be attached to a given

protein.
GPI anchors tether cell surface proteins to the plasma membrane. These
hydrophobic moieties are prepared in the ER, where they are then added to
the nascent protein en bloc. GPI-anchored proteins are often localized to
cholesterol- and sphingolipid-rich lipid rafts, which act as signaling platforms
on the plasma membrane. This type of modification is reversible, as the GPI
anchor can be released from the protein by phosphoinositol-specific
phospholipase C. Indeed, this lipase is used in the detection of GPI-anchored
proteins to release GPI-anchored proteins from membranes for gel separation
and analysis by mass spectrometry.
N-myristoylation is a method to give proteins a hydrophobic handle for
membrane localization. The myristoyl group is a 14-carbon saturated fatty
acid (C14), which gives the protein sufficient hydrophobicity and affinity for
membranes, but not enough to permanently anchor the protein in the
membrane.

N-myristoylation

can

therefore

act

as

conformational

localization switch, in which protein conformational changes influence the

availability of the handle for membrane attachment. Because of this
conditional localization, signal proteins that selectively localize to membrane,
such as Src-family kinases, are N-myristoylated.
N-myristoylation is facilitated specifically by N-myristoyltransferase (NMT)
and uses myristoyl-CoA as the substrate to attach the myristoyl group to the
N-terminal glycine. Because methionine is the N-terminal amino acid of all
eukaryotic proteins, this PTM requires methionine cleavage by the abovementioned MAP prior to addition of the myristoyl group; this represents one
example of multiple PTMs on a single protein.
S-palmitoylation adds a C16 palmitoyl group from palmitoyl-CoA to the
thiolate side chain of cysteine residues via palmitoyl acyl transferases (PATs).

Because of the longer hydrophobic group, this anchor can permanently

anchor the protein to the membrane. This localization can be reversed,
though, by thioesterases that break the link between the protein and the
anchor; thus, S-palmitoylation is used as an on/off switch to regulate
membrane localization. S-palmitoylation is often used to strengthen other
types of lipidation, such as myristoylation or farnesylation (see below). Spalmitoylated proteins also selectively concentrate at lipid rafts (4).
S-prenylation covalently adds a farnesyl (C15) or geranylgeranyl (C20) group
to specific cysteine residues within 5 amino acids from the C-terminus via
farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II). Unlike
S-palmitoylation, S-prenylation is hydrolytically stable. Approximately 2% of
all proteins are prenylated, including all members of the Ras superfamily.
This group of molecular switches is farnesylated, geranylgeranylated or a
combination of both. Additionally, these proteins have specific 4-amino acid
motifs at the C-terminus that determine the type of prenylation at single or
dual cysteines. Prenylation occurs in the ER and is often part of a stepwise
process of PTMs that is followed by proteolytic cleavage by Rce1 and
methylation by isoprenyl cysteine methyltransferase (ICMT).
Proteolysis
Peptide bonds are indefinitely stable under physiological conditions, and
therefore cells require some mechanism to break these bonds. Proteases
comprise a family of enzymes that cleave the peptide bonds of proteins and
are critical in antigen processing, apoptosis, surface protein shedding and
cell signaling.
The family of over 11,000 proteases varies in substrate specificity,
mechanism of peptide cleavage, location in the cell and the length of activity.
While this variation suggests a wide array of functionalities, proteases can
generally be separated into groups based on the type of proteolysis.

Degradative proteolysis is critical to remove unassembled protein subunits

and

misfolded

proteins

and

to

maintain

protein

concentrations

at

homeostatic concentrations by reducing a given protein to the level of small

peptides and single amino acids. Proteases also play a biosynthetic role in
cell biology that includes cleaving signal peptides from nascent proteins and
activating zymogens, which are inactive enzyme precursors that require
cleavage at specific sites for enzyme function. In this respect, proteases act
as molecular switches to regulate enzyme activity.
Proteolysis is a thermodynamically favorable and irreversible reaction.
Therefore, protease activity is tightly regulated to avoid uncontrolled
proteolysis through temporal and/or spatial control mechanisms including
regulation by cleavage in cis or trans and compartmentalization (e.g.,
proteasomes, lysosomes).
The diverse family of proteases can be classified by the site of action, such
as aminopeptidases and carboxypeptidase, which cleave at the amino or
carboxy terminus of a protein, respectively. Another type of classification is
based on the active site groups of a given protease that are involved in
proteolysis. Based on this classification strategy, greater than 90% of known
proteases fall into one of four categories as follows:

Serine proteases

Cysteine proteases

Aspartic acid proteases

Zinc metalloproteases