Sorting, transport and post-translational modifications of proteins

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Protein targeting or protein sorting is the biological mechanism by which proteins are transported to their appropriate destinations within or outside the cell. Proteins can be targeted to the inner space of an organelle, different intracellular membranes, the plasma membrane, or to the exterior of the cell via secretion. Information contained in the protein itself directs this delivery process. Correct sorting is crucial for the cell; errors or dysfunction in sorting have been linked to multiple diseases.

Signal sequence of polypeptide, free and bound ribosomes

Cytosolic proteins are synthesized on free cytosolic ribosomes, while membrane proteins, organelle proteins and proteins released outside the cell are synthesized on ribosomes bound to the rough endoplasmic reticulum (ER). Free and bound ribosomes are structurally and functionally identical, their binding to the ER is determined by the sequence of the synthesized chain. Most proteins destined outside the cytosol have a so-called signal sequence of 13 - 16 amino acids at the N-terminus. Although these sequences vary from protein to protein, the presence of several hydrophobic amino acid residues is characteristic. The sequence is recognized by the SRP particle (signal recognition particle), consisting of six protein subunits and 7SL RNA. It binds to the signal sequence of the synthesized protein and stops translation at the initial stage. The ER membrane contains receptors for SRP. Once the ribosome-SRP complex binds to them, protein synthesis continues with the participation of two other membrane proteins, ribophorin I and II, and at the same time the peptide chain passes through the membrane into the ER cisternae. SRP is again released from the receptor into the cytosol.

The signal sequence is crucial for translocation. If it is genetically engineered to be attached to a cytosolic protein, such as hemoglobin, then this protein is released outside the cell. Peptide translocation is an active membrane process requiring energy (ATP). The translocation is not driven by translation, by the ribosome. Theoretically, it could also occur after the completion of chain synthesis on the free ribosome. However, early binding of the synthesized protein and ribosome to the ER is advantageous and usually necessary, because after synthesis on the free ribosome, the protein could adopt a conformation that would make translocation across the membrane impossible.

The tight space between the translation and translocation sites does not allow the chain to conform before it reaches the other side of the membrane or within it. Some proteins will remain anchored in the membrane, which is also determined by their primary sequence.

In addition to the signal sequence, membrane proteins also have an anchoring, stop-transferase sequence, which terminates translocation across the membrane and the protein remains an anchored part of the membrane. The signal sequence of these proteins can be somewhat distant from the N-terminus. Some even have several such sequences, alternating with stop-transfer sections, so that they are anchored in the membrane in several ways, sometimes multiple times. The signal sequence of secreted proteins is often cleaved by a membrane signalase during translocation. The protein penetrates the ER cisternae. Here, and especially in the Golgi apparatus, it is covalently modified (see Posttranslational glycosylation of proteins) and then transported to its place of function.

Posttranslational glycosylation of proteins

After translocation to the ER cisternae, many proteins are further modified. The signal peptide is cleaved, disulfide bonds are formed. Later, a certain section can be proteolytically cleaved from the polypeptide chain, thereby functionally activating the protein (hormone, enzyme). The function of many proteins can be modified by phosphorylation, acetylation or ADP-ribosylation (p.OOO). Many proteins acquire oligosaccharide residues in the ER and Golgi apparatus, so that they become glycoproteins. The above changes in the finished peptide chain are called post-translational modifications of proteins, or their covalent modifications. This structural and functional maturation of the protein is very important for the regulation of biochemical processes.

Oligosaccharides are bound either by an N-glycosidic bond to an asparagine residue or by an O-glycosidic bond to a serine or threonine residue of a protein. Oligosaccharide precursors are synthesized on an isoprene carrier – dolichol phosphate, contained in the ER membrane. If its phosphate group is on the cytosolic side of the membrane, two N-acetylglucosamines and five mannoses are successively bound to it. Dolichol phosphate with this heptasaccharide is then oriented in the membrane so that the oligosaccharide is on the luminal side of the membrane, directed into the ER cisternae. Here, another four mannoses and three glucoses are transferred to it from another dolichol phosphate precursor.

The thus activated oligosaccharide is transferred to the Asn of the peptide, a phosphatase cleaves off one of the phosphates of dolichol pyrophosphate and the regenerated dolichol phosphate can re-enter the reaction cycle. The aforementioned phosphatase is blocked by the antibiotic bacitracin. The attachment of Glc-NAc to dolichol phosphate is inhibited by the antibiotic tunicamycin.

While still in the ER, three glucoses and one mannose are cleaved from the N-linked oligosaccharide. The protein is then transported to the Golgi apparatus (GA). In its vesicles, oligosaccharides are also bound to the protein by O-glycosidic bonds. N-linked oligosaccharides are further modified. Six mannoses are gradually cleaved and additional GlcNAc, galactoses, fucose and finally sialic acid (N-acetylneuraminic acid) are added. These modifications in the Golgi apparatus are called terminal glycosylation, in contrast to the core glycosylations that take place already in the ER. Oligosaccharides of glycoproteins destined for lysosomes are specifically phosphorylated.

During all processes after the protein is translocated into the ER cisternae, the peptides in the membranes are oriented so that the oligosaccharide residues are on the luminal side of the membrane (in the cisternae, in the GA vesicles, in the transport vesicles). If the transport vesicles fuse with the plasma membrane, the oligosaccharides of the glycoprotein reach the outer, extracellular side of the membrane. A certain asymmetry of the membranes is preserved.

It has been shown that the oligosaccharides of glycoproteins are sometimes a signal, like an address, according to which proteins from the Golgi apparatus are sent to the correct place of their function. There is a mucolipidosis (I-cell disease), the cause of which is a genetic error in the modification of the oligosaccharide residues of lysosomal enzymes. In patients, instead of mannose-6-phosphate, only mannose is present. As a result of this deviation, lysosomal enzymes are not translocated into lysosomes, but outside the cell and can be detected in the blood plasma. In contrast, undecomposed glycosamines and glycolipids accumulate in lysosomes. The patient suffers from psychomotor retardation and skeletal deformities.

However, the glycosylation of most proteins probably has a function other than providing the molecule with a directional signal. Oligosaccharide residues of glycoproteins increase their solubility and help orient the protein molecule towards the aqueous phase. Another role of oligosaccharides is to protect the protein (e.g. immunoglobulin) from the action of proteases. Carbohydrates are a marker for the uptake and subsequent degradation of plasma glycoproteins in the liver. Another significance is seen in the fact that the kinetics of glycoprotein modifications in the ER and GA indicate the step in the passage of these proteins through cellular organelles, thus ensuring the time needed for accurate sorting of synthesized proteins.

Targeting independent of protein glycosylation

Secretory proteins and plasma membrane proteins do not require an oligosaccharide signal for proper localization. Other types of signaling (protein conformation, a certain three-dimensional motif in its structure) are assumed. These proteins may be somehow "sent" to the apical or basolateral part of the plasma membrane, or they are sorted for two types of secretion: constitutive secretion, which is constant and rapid, proteins do not accumulate in secretory vesicles, or directed secretion, when proteins are stored and concentrated in vesicles and released from the cell only upon hormonal stimulation. Only then do the vesicles fuse with the cytoplasmic membrane and their contents are released outside the cell. Examples of directed secretion are the release of digestive enzymes from pancreatic acinar cells or the release of peptide hormones from endocrine cells.

Targeting mitochondrial proteins

Most mitochondrial proteins are synthesized on free cytosolic ribosomes and post-translationally incorporated into mitochondria. Some proteins are intended for the outer, others for the inner mitochondrial membrane, others for the intermembrane space and for the matrix. The localization of the protein is determined by the sequence of the N-terminal part of the chain, the so-called mitochondrial entry sequence, which is rich in basic amino acid residues and serine and threonine. If the protein is to be anchored in the outer mitochondrial membrane, then the entry sequence is followed by an anchoring sequence and a second positively charged section.

A proton transmembrane gradient is required for the protein to pass through the inner mitochondrial membrane. Passage through the outer membrane does not require this energy source. The entry sequence is usually proteolytically cleaved after passing through the inner (not outer) membrane.

A protein transferred from the cytosol to the matrix first binds its presequence to a receptor on the outer mitochondrial membrane. At the site of translocation, the outer and inner membranes abut each other and the protein passes through both at once. In the matrix, the transferred protein is cleaved from the membrane-anchored presequence.

Intermembrane proteins (e.g. cytochrome b) are first anchored in the inner mitochondrial membrane and then cleaved from the intermembrane space by a special protease. Some intermembrane proteins (cytochrome c) remain bound to the inner membrane.

During the passage through the membrane, mitochondrial proteins are fully unfolded and then restore their tertiary structure.

Bacteria also distribute synthesized proteins using signal sequences. Some of their proteins are destined for the plasma membrane, others for the outer membrane, others for the periplasmic space, or are rarely released outside the cell. The translocation is driven by a proton gradient. The analogy with mitochondrial targeting is therefore obvious.

Targeting nuclear proteins

Nuclear proteins (histones, polymerases, etc.) are synthesized on free cytosolic ribosomes. The nucleus then enters through pores in the nuclear membrane. These pores open upon a special signal, the nature of which is still mostly unknown. In the case of the T-antigen of the SV40 virus, an amino acid sequence was discovered between positions 127 and 131 that is crucial for the protein to enter the nucleus (nuclear localization sequence). A single amino acid change in this region prevents the protein from leaving the cytosol. If this sequence is experimentally inserted into the structure of another protein, this protein appears in the nucleus even though it is not a nuclear protein.

Decision-making mechanism for the destruction of non-functional proteins

Proteins intended for destruction and removal from the cell are also subject to specific targeting. The biological half-life of cytosolic proteins is very variable, from several minutes to more than twenty hours. The duration of the existence of these proteins is determined by their N-terminal amino acid. Met, Gly, Ala, Ser, Thr and Val are the amino acid No. 1 of more stable proteins (half-life longer than 20 hours). N-terminal Ile or Glu signal about half an hour of peptide survival. Pro, Leu, Phe, Asp, Lys and Arg ensure a half-life of only a few minutes. Such a short half-life is important for regulatory peptides, e.g. hormones, so that changes in regulation are sufficiently rapid. This signaling arose in the early phase of the development of life, as it is known in bacteria, yeast and mammals. The mechanism of the described targeting is not fully understood. An important role is played by the protein ubiquitin (Mr=8500), which is present in all eukaryotic cells. The C-terminal Gly of ubiquitin is covalently linked to the ε-NH2 lysine of the protein to be degraded. Interestingly, ubiquitin is first activated by ATP and three enzymes, and is bound to their -SH groups. This activation is thus reminiscent of fatty acid activation or aa-tRNA synthesis (amino acid activation), which is one example of a general principle that we encounter more often in biochemistry.

Receptor-mediated endocytosis

The previous chapters dealt with the targeting of intracellular proteins. However, the principle of targeting also applies to the uptake of proteins from the extracellular space by endocytosis, which is mediated by the interaction of the protein with a membrane receptor on the cell surface.

Receptor

The receptor in question is a glycoprotein located in special membrane locations, the so-called coated pits. On the cytosolic side of these locations is a clathrin coat. Thanks to its three-armed structure, clathrin is able to form a mesh-like coat around the membrane pits or around various cytoplasmic vesicles, vacuoles. After the supply of ATP, the clathrin network can be enzymatically disrupted and clathrin can be used for further interactions.

After the engulfed protein binds to the receptor, the pit deepens, and clathrin eventually forms a closed network, so that a coated vesicle is released from the membrane into the cytoplasm. This then quickly loses its clathrin coat and turns into an endosome or receptosome. It usually enlarges by fusing with other endosomes. The function of these organelles is to decide where the engulfed protein should be transported. An important mechanism is the acidification of the endosome contents, which occurs through the activity of the ATP-dependent H+/K+ pump in the endosome membrane.

Proteins after ingestion

Other proteins taken up by receptor-mediated endocytosis have a different fate in endosomes. LDL-apoprotein, which carries cholesterol, binds to a membrane receptor and is transferred to the lysosome after endocytosis. Here it is degraded by lysosomal proteases, while the receptor is reused on the cell surface. Immune complexes, insulin or some growth factors are degraded in lysosomes along with their receptors. This is an example of modulation of the effect of protein hormones, as this reduces their concentration in the blood and the number of receptors in target cells.

Sources

Protein targeting. Online. In: Wikipedia: the free encyclopedia. San Francisco (CA): Wikimedia Foundation, 2001-. Dostupné z: https://en.wikipedia.org/wiki/Protein_targeting. [cit. 2025-05-30].

Posttranslační úpravy a targeting proteinů. Online. Wikiskripta. 2020. Dostupné z: https://www.wikiskripta.eu/w/Posttransla%C4%8Dn%C3%AD_%C3%BApravy_a_targeting_protein%C5%AF. [cit. 2025-05-30].