Post-translational modifications and protein targeting

In both bacteria and eukaryotes, some proteins are destined for the cytosol, others are part of the membranes, or are secreted out of the cell.In eukaryotic cells, some proteins are sent to cellular organelles after completion of their synthesis, such as lysosomes, mitochondria, of the cell nucleus and in plants to chloroplasts. Sending new proteins to their destination is called targeting (angl. target = destination).

Polypeptide signal sequences, 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 exactly the same, their binding to the ER is determined by the sequence of the synthesized chain. Most proteins determined outside the cytosol have a so-called 13-16 amino acid signal sequence 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 a 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 an early stage. The ER membrane contains receptors for SRP. Once the ribosome-SRP complex binds to them, proteosynthesis continues with the participation of two other membrane proteins, riboforin I and II, and at the same time the peptide chain passes through the membrane into the ER tank. The SRP is again released from the receptor into the cytosol. The signal sequence is crucial for translocation. If it is attached to a cytosolic protein, such as hemoglobin,by gene manipulation, then this protein is released outside the cell. Peptide translocation is an active energy-requiring membrane process (ATP). Permeation is not driven by translation,the ribosome. Theoretically, it could take place even after the synthesis of the free ribosome chain has been completed. However, early binding of the synthesized protein and ribosome to the ER is advantageous and mostly necessary because, after synthesis on the free ribosome, the protein could assume a conformation that would prevent translocation across the membrane.

The tight space between the translation site and the translocation will not allow the chain to conform before or on the other side of the membrane. Some proteins remain anchored in the membrane, which is also due to their primary sequence.

In fact, 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 may also 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 several times (see figure). The signal sequence of the secreted proteins is still cleaved by the membrane signalase during translocation. The protein penetrates the ER tank. Here and especially in the Golgi apparatus, it is covalently modified (see Post-translational glycosylation of proteins) and then transported to the site of its function.

Post-translational glycosylation of proteins
After translocation into ER cisterns, many proteins are further modified.The signal peptide is cleaved, disulfide bonds are formed. Later, a certain section can be cleaved from the polypeptide chain proteolytically, and thus the protein is functionally activated (hormone, enzyme). The function of a number of proteins can be modified by phosphorylation, acetylation or ADP-ribosylation (p.OOO). Many proteins acquire oligosaccharide residues in the ER and Golgi, making them glycoproteins.These changes in the finished peptide chain are called post-translational modifications of proteins, or also their covalent modifications. This structural and functional maturation of the protein is very important for the regulation of biochemical processes.

Oligosaccharides bind by either an N-glycoside bond to an asparagine residue or an O-glycoside bond to a serine or threonine residue of a protein. Oligosaccharide precursors are synthesized on an isoprene support – 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 sequentially bind to it. The dolichol phosphate with this heptasaccharide is then oriented in the membrane so that the oligosaccharide is on the luminary side of the membrane, heading to the ER tank. Here, four more mannose and three glucose are transferred to it from another dolicholphosphal precursor (see figure).

The oligosaccharide thus activated is transferred to the Asn peptide, the phosphatase cleaves one of the dolicholpyrophosphate phosphates and the regenerated dolichol phosphate can re-enter the reaction cycle. The antibiotics bacitracin blocks this phosphatase.The attachment of Glc-NAc to dolichol phosphate is inhibited by the antibiotic tunicamycin.

In the ER, three glucose and one mannose are cleaved from the N-linked oligosaccharide. The protein is then transferred to the Golgi apparatus (GA).In its vesicles, oligosaccharides also bind to the protein through an O-glycoside bond. The N-linked oligosaccharides are further modified. Six mannoses are sequentially cleaved and additional GlcNAc, galactose, fucose and finally sialic acid (N-acetylneuraminic acid) are added. These modifications in the Golgi apparatus are called terminal glycosylations, in contrast to the basic glycosylations (core glycosylations) already taking place in the ER. Lysosome-derived glycoprotein oligosaccharides are specifically phosphorylated.

During all processes after transfer of the protein to the ER tanks, the peptides in the membranes are oriented so that the oligosaccharide residues are on the luminal side of the membrane (in the tank, in the GA vesicles, in the transport vesicles). When the transport vesicles merge with the plasma membrane, the glycoprotein oligosaccharides reach the outer, extracellular side of the membrane. Some asymmetry of the membranes is maintained.

Glycoprotein oligosaccharides have sometimes been shown to signal the address, at which proteins from the Golgi apparatus are sent to their proper function. There is mucolipidosis (I-cell disease), which is caused by a genetic error in the modification of oligosaccharide residues of lysosomal enzymes. In patients, instead of mannose-6-phosphate, there is only mannose. As a result of this variation, lysosomal enzymes are not transferred to lysosomes, but out of the cell and can be detected in blood plasma. In contrast, undecomposed glycosamines and glycolipids accumulate in lysosomes. The patient suffers from psychomotor retardation and skeletal deformities.

However, glycosylation of most proteins probably has a different function than providing the molecule with a targeting 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 (eg. 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 ER and GA indicate a step in the passage of these proteins through cellular organelles, thus ensuring the time required for accurate sorting of synthesized proteins.

Targeting independent of protein glycosylation
Plasma membrane secretory proteins and proteins do not need an oligosaccharide signal to properly localize. Other types of signaling are assumed (conformation of the protein, a certain three-dimensional motif in its structure). These proteins can be "sent" to the apical or basolateral part of the plasma membrane in some way, or they are classified for two types of secretion: constitutive secretion, which is constant and rapid, proteins do not condense in secretory vesicles, or controlled secretion, when proteins are stored and they concentrate in the vesicles and are released from the cell on a hormonal impulse. Only then do the vesicles merge with the cytoplasmic membrane and their contents are released outside the cell. Examples of controlled secretion are the release of digestive enzymes from acinar cells of pancreas or the release of peptide hormones from endocrine cells.

Targeting of mitochondrial proteins
Most mitochondrial proteins are synthesized on free cytosolic ribosome and posttranslationally 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 region of the chain, the so-called mitochondrial input sequence, which is rich in basic amino acid residues and serine and threonine. If the protein is to anchor in the outer mitochondrial membrane, then the input sequence is followed by an anchoring sequence and a second positively charged region.

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 input sequence is proteolytically cleaved after passing through the inner (not outer) membrane. The protein transferred from the cytosol to the matrix first binds by its presequence to the receptor on the outer mitochondrial membrane. At the site of permeation, the outer and inner membranes abut each other and the protein permeates both at the same time. In the matrix, the transferred protein is cleaved from the membrane-anchored presequence.

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

During passage through the membrane, mitochondrial proteins fully develop and then restore the 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 thus obvious.

Targeting of nuclear proteins
Nuclear proteins (histones, polymerasy a j.) are synthesized on free cytosolic ribosomes.The pores in the nuclear membrane then enter the nucleus. These pores open to a special signal, the nature of which is usually unknown. In the case of the SV40 virus T-antigen, an amino acid sequence was found between 127 and 131, which is crucial for the entry of the protein into the nucleus (nuclear localization sequence). Replacing a single amino acid in this region prevents the protein from leaving the cytosol. If this sequence is experimentally inserted into the structure of another protein, this protein will appear in the nucleus even if it is not a nuclear protein.