15. Posttranslational modifications and protein transport.

In the final stage of protein synthesis, the nascent polypeptide chain is folded
and processed into its biologically active form. During or after its synthesis,
the polypeptide progressively assumes its native conformation, with the
formation of appropriate hydrogen bonds and van der Waals, ionic, and
hydrophobic interactions. The linear, or one-dimensional, genetic message in
the mRNA is converted into the three dimensional structure of the protein.
Some proteins, (pro- and eukaryotic) do not attain their final biologically
active conformation until they have been altered by one or more processing
reactions called post-translational modifications.
-Protein phosphorylation involves the enzyme-catalyzed transfer of the
terminal phosphate group of an ATP molecule to the hydroxyl group on a
serine, threonine, or tyrosine side chain of the protein. A protein kinase
catalyzes this reaction, and the reaction is essentially unidirectional because of
the large amount of free energy released when the phosphate–phosphate bond
in ATP is broken to produce ADP. A protein phosphatase catalyzes the
reverse reaction of phosphate removal, or dephosphorylation. Cells contain
hundreds of different protein kinases, each responsible for phosphorylating a
different protein or set of proteins. There are also many different protein
phosphatases; some are highly specific and remove phosphate groups from
only one or a few proteins, whereas others act on a broad range of proteins and
are targeted to specific substrates by regulatory subunits. The state of
phosphorylation of a protein at any moment, and thus its activity, depends on
the relative activities of the protein kinases and phosphatases that modify it.
The functional significance of this modification varies from one protein to the
next.

-Extra carboxyl groups may be added to Glu residues of some proteins. For
example, the blood-clotting protein prothrombin contains a number of
g-carboxyglutamate residues in its amino-terminal region, introduced by an
enzyme that requires vitamin K. These carboxyl groups bind Ca2+, which is
required to initiate the clotting mechanism.

-Addition of a methyl group to lysine or arginine residues. Methylation can

affect gene expression, protein-protein interactions, and enzyme activity.
Histone methylation, for example, is involved in chromatin remodeling.
For example, monomethyl- and dimethyllysine residues occur in some muscle
proteins and in cytochrome c. The calmodulin of most species contains one
trimethyllysine residue at a specific position. In other proteins, the carboxyl
groups of some Glu residues undergo methylation, removing their negative
charge. The first residue inserted in all polypeptides is N- formylmethionine
(in bacteria) or methionine (in eukaryotes). However, the formyl group, the
amino terminal Met residue, and often additional amino-terminal (and, in
some cases, carboxyl-terminal) residues may be removed enzymatically in
formation of the final functional protein.

-The carbohydrate side chains of glycoproteins are attached covalently during

or after synthesis of the polypeptide. In some glycoproteins, the carbohydrate
side chain is attached enzymatically to Asn residues (N-linked
oligosaccharides), in others to Ser or Thr residues (O- linked
oligosaccharides). Many proteins that function extracellularly, as well as the
lubricating proteoglycans that coat mucous membranes, contain
oligosaccharide side chains.

-A number of eukaryotic proteins are modified by the addition of groups

derived from isoprene (isoprenyl groups). A thioether bond is formed between
the isoprenyl group and a Cys residue of the protein. The isoprenyl groups are
derived from pyrophosphorylated intermediates of the cholesterol
biosynthetic pathway, such as farnesyl pyrophosphate. Proteins modified in
this way include the Ras proteins, products of the ras oncogenes and proto-
oncogenes, and G proteins, and lamins, proteins found in the nuclear matrix.
The isoprenyl group helps to anchor the protein in a membrane. The
transforming activity of the Ras oncogene is lost when isoprenylation of the
Ras protein is blocked, a finding that has stimulated interest in identifying
inhibitors of this post-translational modification pathway for use in cancer
therapy.

-Many proteins are initially synthesized as large, inactive precursor

polypeptides that are proteolytically trimmed to form their smaller, active
forms–proinsulin, some viral proteins, and proteases such as pepsinogen,
chymotrypsinogen and trypsinogen. After folding into their native
conformations, some proteins form intrachain or interchain disulfide bridges
between Cys residues. In eukaryotes, disulfide bonds are common in proteins
to be exported from cells. The cross-links formed in this way help to protect
the native conformation of the protein molecule from denaturation in the
extracellular environment.

-Although a protein chain can fold into its correct conformation without out-
side help, in a living cell special proteins called molecular chaperones
often assist in protein folding. Many molecular chaperones are called
heat-shock proteins (designated hsp), because they are synthesized in
dramatically increased amounts after a brief exposure of cells to an elevated
temperature. This reflects the operation of a feedback system that responds to
an increase in misfolded proteins (such as those produced by elevated
temperatures) by boosting the synthesis of the chaperones that help these
proteins refold. There are several major families of molecular chaperones,
including the hsp60 and hsp70 proteins. The hsp60 and hsp70 proteins each
work with their own small set of associated proteins when they help other
proteins to fold. These hsps share an affinity for the exposed hydrophobic
patches on incompletely folded proteins, and they hydrolyze ATP, often
binding and releasing their protein substrate with each cycle of ATP
hydrolysis. In other respects, the two types of hsp proteins function
differently. The hsp70 machinery acts early in the life of many proteins (often
before the protein leaves the ribosome), with each monomer of hsp70 binding
to a string of about four or five hydrophobic amino acids . On binding ATP,
hsp70 releases the protein into solution allowing it a chance to re-fold.

In contrast, hsp60-like proteins form a large barrel-shaped structure that acts

after a protein has been fully synthesized. This type of chaperone, sometimes
called a chaperonin, forms an “isolation chamber” for the folding process. To
enter a chamber, a substrate protein is first captured via the hydrophobic
entrance to the chamber. The protein is then released into the interior of the
chamber, which is lined with hydrophilic surfaces, and the chamber is sealed
with a lid, a step requiring ATP. Here, the substrate is allowed to fold into its
final conformation in isolation, where there are no other proteins with which
to aggre- gate. When ATP is hydrolyzed, the lid pops off, and the substrate
protein, whether folded or not, is released from the chamber.In the case of
protein folding, ATP hydrolysis allows chaperones to recognize a wide variety
of misfolded structures, to halt any further misfolding, and to recommence the
folding of a protein in an orderly way.

Protein transport involves the movement of a protein from one cellular or

extracellular compartment to another. It can be facilitated by a variety of
pathways including membrane trafficking, protein translocation, and
endocytosis or exocytosis. The transport of proteins between cellular
compartments is highly regulated, and signal peptides play a crucial role in
guiding proteins to their appropriate destinations. Signal peptides are short
amino acid sequences located at the N-terminus of newly synthesized
polypeptide chains. These sequences serve as targeting signals, guiding the
proteins to their correct subcellular locations.

-In nuclear transport, proteins destined for the nucleus typically contain a
specific amino acid sequence known as a nuclear localization signal (NLS).
This signal is often rich in basic amino acids, including positively charged
arginine (Arg) and lysine (Lys). The NLS is recognized by nuclear transport
receptors, such as importins, which facilitate the transport of the protein
through the nuclear pore complex.
Nuclear Pore Complexes (NPCs) are large protein complexes embedded in the
nuclear envelope, which is the double membrane that surrounds the nucleus.
These complexes serve as selective gates that actively regulate the transport of
macromolecules between the nucleus and the cytoplasm. NPCs have a central
channel that allows the passage of smaller molecules but restricts the passage
of larger macromolecules. The nuclear envelope effectively separates the
nuclear and cytoplasmic compartments, ensuring the proper functioning and
organization of cellular processes. Nuclear transport occurs through a process
known as gated transport. The nuclear envelope acts as a barrier, and
transport through the nuclear pores is selective and regulated. Importins
and exportins are key players in gated transport. Importins facilitate the
import of proteins into the nucleus, while exportins mediate the export of
proteins from the nucleus to the cytoplasm. Proteins with an NLS are
recognized by importin proteins in the cytoplasm. The importin-protein
complex enters the nucleus through the nuclear pore. Once inside the nucleus,
the complex undergoes disassembly, releasing the protein cargo. Proteins that
need to be exported from the nucleus contain a nuclear export signal (NES).
Exportins bind to proteins with NES in the nucleus, forming a complex. The
exportin-protein complex is transported through the nuclear pore to the
cytoplasm. In the cytoplasm, the complex dissociates, and the protein is
released.

-In mitochondrial transport, proteins targeted to mitochondria typically

contain a mitochondrial targeting signal, often referred to as a mitochondrial
presequence or transit peptide. The mitochondrial presequence is typically
located at the N-terminus and is rich in positively charged amino acids. The
mitochondrial targeting signal is recognized by receptors on the outer
mitochondrial membrane. Import receptors, such as Tom20 and Tom22,
interact with the targeting signal. The protein-receptor complex is transported
through the Translocase of the Outer Membrane (TOM) complex, which forms
a channel in the outer mitochondrial membrane. The TOM complex
recognizes the targeting signal and facilitates the translocation of the protein
into the intermembrane space. Proteins continue to move through the
Translocase of the Inner Membrane (TIM) complex, which is responsible for
translocating proteins across the inner mitochondrial membrane. The
mitochondrial targeting signal is often cleaved during or after translocation,
yielding the mature protein. Proteins imported into the mitochondria often
undergo folding and assembly processes to attain their functional
conformation. Chaperone proteins within the mitochondria assist in the
folding and assembly of imported proteins.

-Protein transport to the endoplasmic reticulum (ER) involves a

co-translational process, where proteins are translocated into the ER as they
are being synthesized on ribosomes. This process is crucial for the proper
folding, modification, and trafficking of proteins. Proteins targeted to the ER
have a specific amino acid sequence known as a signal peptide or signal
sequence. The signal peptide is typically 5-10 hydrophobic amino acids and is
located at the N-terminus of the polypeptide chain. As the nascent polypeptide
chain emerges from the ribosome during translation, the signal peptide is
recognized by a signal recognition particle (SRP). The SRP binds to the signal
peptide and temporarily halts translation. The SRP-ribosome complex is
directed to the ER membrane, where it interacts with the SRP receptor on the
ER membrane. The SRP is then released, and translation resumes. The
nascent polypeptide chain is translocated across the ER membrane
simultaneously with its synthesis, a process known as cotranslational
translocation. The translocon, a protein channel embedded in the ER
membrane, facilitates the translocation of the growing polypeptide chain into
the ER lumen or membrane. The signal peptide plays a crucial role in guiding
the polypeptide through the translocon. Once the protein is completely
translocated, the signal peptide is cleaved by a signal peptidase, resulting in
the removal of the signal sequence. The cleavage occurs on the luminal side of
the ER membrane. Proteins within the ER undergo proper folding and
post-translational modifications with the assistance of molecular chaperones
and enzymes. Protein disulfide isomerases help form and rearrange disulfide
bonds. The ER has a quality control system to ensure that only properly folded
and assembled proteins are allowed to exit. Misfolded or unassembled
proteins may be targeted for degradation through a process called
ER-associated degradation (ERAD). Many proteins translocated into the ER
undergo glycosylation, where oligosaccharide chains are added to specific
asparagine residues. Other post-translational modifications, such as disulfide
bond formation, may also occur in the ER. Once proteins are properly folded
and modified, they may be transported to the Golgi apparatus for further
processing and sorting.

-The transport of proteins through the Golgi involves the formation of

vesicles that bud off from one Golgi cisterna and fuse with the next
cisterna.Vesicular transport through the Golgi occurs through a series of
consecutive steps: bud formation, vesicle docking to the target compartment,
and membrane fusion. Transport intermediates may include small, spherical
transport vesicles or larger, irregularly shaped fragments of Golgi cisternae.
These intermediates aid in the movement of cargo proteins through the Golgi
stacks.
Glycosylation is a crucial modification that occurs in the Golgi apparatus.
Addition and removal of oligosaccharides to proteins take place, influencing
their structure and function.
Proteins that fail to fold correctly or do not meet quality control standards in
the Golgi may be partially degraded before being targeted for secretion or
lysosomal delivery. Proteins in the Golgi are sorted based on specific signals
and modifications. Different Golgi cisternae are associated with distinct
enzymatic activities, allowing for the sequential modification of proteins.
Some proteins are directed to the cell membrane for constitutive or regulated
secretion. Constitutive secretion involves spontaneous fusion of transport
vesicles with the cell membrane, releasing the proteins into the extracellular
space. Regulatory secretion involves fusion triggered by specific signals.
Vesicles may be coated with clathrin during this process.
Proteins targeted for lysosomes are modified with Mannose-6-phosphate
(M6P) as a label. M6P serves as a recognition signal for sorting proteins into
vesicles

Membrane trafficking is the process by which proteins and other

macromolecules are distributed throughout the cell, and released to or
internalized from the extracellular space (endo- and exocytosis). It uses
membrane-bound vesicles as transport intermediaries. Cargo molecules are
enclosed within or associated with the membrane of these vesicles. These
transport mechanisms are responsible for maintaining the proper function of a
cell. On the other hand, membrane trafficking defects can cause severe or fatal
diseases. Charcot-Marie-Tooth disease and Centronuclear Myopathy are
associated with defects in clathrin-mediated endocytosis, which may affect
membrane trafficking processes.