Signal Peptides Nuclear Proteins Mitochondrial Proteins Chloroplast Proteins Rough Endoplasmic Reticulum Organelle Proteins Exported Proteins Transmembrane Proteins Signal Recognition Particles SRP receptors Signal Peptidase Signal Peptide Search Engine
Signal Peptides
Nuclear Proteins
Mitochondrial Proteins
Chloroplast Proteins
Rough Endoplasmic Reticulum
Organelle Proteins
Exported Proteins
Transmembrane Proteins
Signal Recognition Particles
SRP receptors
Signal Peptidase
Signal Peptide Search Engine
Signal peptides The properties of the amino acids that constitute the signal peptide region of a protein are the significant factors determining interaction with the protein transport system, hence the destination to which that protein is delivered. Different classes of signal peptide are used to specify different cellular placement. It should be reiterated that not all proteins possess signalling regions; those which don't are maintained in the cytoplasm. The common structure of signal peptides from various proteins is commonly described as a positively charged n-region, followed by a hydrophobic h-region and a neutral but polar c-region. The (-3,-1)-rule states that the residues at positions -3 and -1 (relative to the cleavage site) must be small and neutral for cleavage to occur correctly.
In many instances the amino acids comprising the signal peptide are cleaved off the protein once its final destination has been reached. The cleavage is catalysed by enzymes known as signal peptidases. Exceptions to this rule do exist (see below).
Ovalbumin 1ova (1.0Mb) [Bbk|BNL|ExP|Waw|Hal] is an example of a secretory protein which does not naturally have its signal sequence cleaved. The 100 N-terminal residues are found to be necessary for transport through the membrane to be effected.
Different organelles have adopted subtle variations on the general theme of signal peptide targeting of proteins:-
All nuclear proteins are synthesised on free ribosomes in the cytoplasm. Proteins destined for the nucleus have to negotiate the nuclear membrane; a double membrane. Unlike the situation elsewhere these proteins are able to cross into the nucleus from the cytoplasm (and vice versa) whilst still folded. The reason for this is due largely to the existence of specialised nuclear pores, which govern the transposition process, and involve the direct expenditure of energy. Protein coated gold beads have been instrumental in demonstrating the selectivity of the nuclear pore complexes. Comparison of a large number of nuclear proteins shows the presence of a short sequence of amino acids specifying nuclear import, although quite different sequences are utilised by different proteins. These nuclear localisation signals may be at the N-terminal or C-terminal ends of proteins.
Another unusual and important feature of nuclear proteins is that nearly all mature functional molecules still possess their signal peptides, i.e. there is no cleavage of this signal region upon importation into the nucleus. The reason for this becomes clear when one understands the processes that accompany cell division; during both mitosis and meiosis the nuclear envelope is completely (in higher eukaryotes) or partially (in lower eukaryotes) dissolved to allow proper segregation of the cellular contents, including the chromosomes. All nuclear proteins are exposed to the cytoplasm. However, once the nuclear membrane reforms around the chromosomes, these same proteins are redirected to the new nucleus because they still possess the appropriate signal peptides. If the signals had been removed then those proteins would not be shuttled back to the nucleus.
Some proteins are prevented from entering the nucleus immediately following their synthesis by masking the nuclear localisation signal. This can be achieved via either chemical modification of the signal, or by interaction with inhibitory cytosolic proteins.
Mitochondral Proteins
Mitochondria are double membrane bound organelles involved in the production of energy. The internal membrane relies on an electrical potential to drive protein translocation. Mitochondrial proteins (with the exception of those that are produced by the organelles own ribosomes) are made by cytosolic free ribosomes and imported post-translationally by receptors which reside at points of contact between the inner and outer membranes. Most of these proteins are present in the cytoplasm as precursors of the active forms. In contrast to nuclear proteins, those targeted across the mitochondrial membranes are only able to do so in an unfolded state. Energy provided by the hydrolysis of ATP is required for all transfers.
Some cytoplasmic proteins, such as cytochrome C peroxidase, are directed to the intermembrane space, while others (porin) are placed in the outer membrane, and yet others (cytochrome C oxidase) are sent to the inner membrane. Most are directed to the matrix of the mitochondrion. The following experiments have provided support.
Some proteins possess an additional 20 - 60 amino acids not present on the mature proteins. This region is known as the uptake-targeting sequence. The general sequence of events is as follows;
Cytosolic proteins, that have an uptake-targeting sequence, combine with unfolding factors in the cytoplasm, that disrupt higher levels of protein folding > Binding to receptors on the outer membrane of the mitochondrion occurs > Membrane translocation to the transport channel proteins > Passage of the protein through the double membrane > Cleavage of the uptake-targeting sequence by matrix protease > Refolding of the mature protein. The uptake-targeting sequence can be a matrix-targeting sequence, that is located on the N-terminus of proteins. This sequence contains all the information necessary to target a protein from the cytosol to the matrix of mitochondria. A simple experiment is able to demonstrate the effectiveness of these sequences. As with nuclear proteins, a comparison of different uptake-targeting sequences reveals no homology, but does show that they all possess common characteristics (rich in positively charged amino acids, arginine and lysine, and hydroxyl bearing ones, serine and threonine). Receptors on the surface of the mitochondria show affinity for these physical characteristics.
Cytosolic proteins, that have an uptake-targeting sequence, combine with unfolding factors in the cytoplasm, that disrupt higher levels of protein folding > Binding to receptors on the outer membrane of the mitochondrion occurs > Membrane translocation to the transport channel proteins > Passage of the protein through the double membrane > Cleavage of the uptake-targeting sequence by matrix protease > Refolding of the mature protein.
The uptake-targeting sequence can be a matrix-targeting sequence, that is located on the N-terminus of proteins. This sequence contains all the information necessary to target a protein from the cytosol to the matrix of mitochondria. A simple experiment is able to demonstrate the effectiveness of these sequences.
As with nuclear proteins, a comparison of different uptake-targeting sequences reveals no homology, but does show that they all possess common characteristics (rich in positively charged amino acids, arginine and lysine, and hydroxyl bearing ones, serine and threonine). Receptors on the surface of the mitochondria show affinity for these physical characteristics.
Other proteins have two N-terminal uptake-targeting sequences on the precursors. These proteins tend to be targeted to regions of the mitochondria other than the matrix.
The order of events leads the protein to the matrix initially, where the first signal (a matrix-targeting sequence) is removed. Then the protein is redirected across a further membrane to another compartment where the second targeting sequence is cleaved. Cytochrome b2 is a good example. The final destination of this enzyme is the intermembrane space. Cytochrome b2 precursors enter the matrix before transfer to the intermembrane space. The effects of mutating these sequences are predictable.
All mitochondrial proteins that are imported have at least one signal peptide. However, not all are removed. Porin is a good example of one that retains its signal peptide.
Chloroplasts are plant organelles, which are surrounded by a double membrane, and also have an additional internal membrane (the thylakoid). They resemble the mitochondrion with reference to protein import, in that energy expenditure is required to import unfolded proteins by specialised receptors. Unlike mitochondrion ATP hydrolysis is the driving force for protein trafficking within the organelle, instead of membrane electrical potential . Chloroplast ribosomes contribute a small fraction of the organelles proteins, the rest are imported. Once again, all imported proteins are directed to the stroma (equivalent to mitochondrial matrix) first, by the presence of a stromal import sequence. Proteins which function in other compartments carry an additional targeting sequence.
Rough Endoplasmic Reticulum (rER)
Proteins that are exported from the cell, as well as those delivered to specialised organelles and retained in the closed lumen of the ER, have to pass through the rER membrane. To ensure that these proteins are not released into the cytosol (where they can have grave consequences for the cell) the trafficking machinery has certain safeguards. Proteins possessing certain signal peptides are quickly recognised soon after their translation is begun. The ribosomes encoding these classes of polypeptides are then directed to the outer membrane of the rER to conclude protein synthesis in a highly controlled manner (a process known as cotranslationally). The completed protein is either placed in the lumen of the rER, or becomes embedded in it, but never released into the cytosol. Signal recognition particles and their receptors play a central part in this process. A protein handled by this pathway has a number of final destinations; each is discussed below.
(Some exceptions to the above rule exist. Several yeast proteins which enter the ER post-translationally require the presence of factors able to adhere to the protein and disrupt its folding, with the added need for ATP hydrolysis.)
Endoplasmic Reticulum Lumen The protein may be retained in the lumen of the ER. A signal consisting of a short, four amino residue, known as the ER retention signal, is required. The signal found at the C-terminal of a resident ER lumen protein, BiP, is Lys-Asp-Glu-Leu. This sequence does not anchor the protein to the ER membrane, instead it causes any molecules detected in the Golgi to be cycled back. Experiments reveal the potency of this small signal. Organelle Proteins With the exception of mitochondria, chloroplasts, nuclei, and peroxisomes, all other organelles receive their proteins via the rER. Many proteins are modified by the Golgi apparatus prior to delivery. Additional signals on the protein must direct the delivery machinery to place it in the correct location. Certain proteins not only have signal peptides but also possess signal patches, e.g. lysosomal hydrolases, where the patch allows proper modification of the hydrolase by special phosphotransferases. It is not unusual to observe C-terminal signal peptides. Exported Proteins All cells possess a constitutive secretory pathway, where proteins destined for export are secreted from the cell. These proteins pass through the ER-Golgi processing pathway where modifications may occur. If no further signals are detected on the protein it will be directed to the cells surface for secretion. Some proteins are stored prior to release; these do have additional signals, and utilise the regulated secretory pathway. Transmembrane Proteins Proteins can end up as integral membrane components. These proteins are said to be transmembrane, and initially follow the same pathway as those which pass through to the ER lumen, except that they are retained in the ER membrane by the presence of stop-transfer signals. Stop-transfer signals are hydrophobic stretches of about 20 amino acid residues that adopt an alpha-helical conformation as they transverse the membrane. Membrane embedded proteins are anchored in the phospholipid bilayer. The membrane can be distributed to different locations in the cell including the plasma membrane and organelle membranes. These membranes have a "sideness" and cannot flip over, i.e. a protein with its N-terminal facing the ER lumen will never face the cytosol. Single membrane spanning proteins generally have a hydrophobic region that anchors that sequence in the phospholipid bilayer via a alpha-helix configuration. Proteins of this type can become embedded in the membrane in three ways:- In the simplest class, the N-terminal region has a signal peptide that passes through the membrane and is cleaved upon exiting into the lumen of the ER. However, a hydrophobic region in the central part of the polypeptide acts as a stop-transfer sequence. This leaves the C-terminal tail protruding from the cytosolic face of the ER. In another class, the signal peptide is internal on the polypeptide chain and is not cleaved. It acts as a start-transfer signal, initiating protein translocation, but upon release from the SRP receptor, anchors the peptide in the membrane. The only difference between this class and the last is the orientation of the signal peptide. It is reversed and results in the C-terminal of the polypeptide being directed into the ER lumen and the N-terminal in the cytoplasm. Multiple membrane spanning proteins result from the polypeptide chain passing back and forth across the phospholipid bilayer. Normally, the first helical domain is an internal uncleaved signal-start sequence that commences the transfer of the protein. The second helical region acts as a stop-transfer signal and prevents further transfer of the polypeptide. The result is the insertion of a hairpin loop across the membrane. Interaction with the SRP and a SRP receptor are necessary. Subsequent helical domains alternate as either start-transfer signal sequences or stop-transfer sequences. Each pair introduces another hairpin loop. Interestingly, SRP and SRP receptors are not involved. There are many examples of proteins of this type. A large class are known as heptahelicals, because they represent peptides that transverse the membrane seven times. The opsin class of proteins, which combine with a chromophore to generate the light stimulated molecules in the photoreceptors of animals, are a classic example. In this example (human cone opsin) of the hypothetical two-dimensional representation of the human long-wavelength or middle-wavelength sensitive opsin molecule, each of the seven transmembrane helices forms a alpha-helix as it passes through the phospholipid membrane (yellow blocks). The first helix has the characteristics of a start-transfer signal peptide, while the second helix is also a start-transfer signal and the third helix is a stop-transfer signal. The remaining four helical domains would alternate between start-transfer and stop-transfer signals.
Endoplasmic Reticulum Lumen
The protein may be retained in the lumen of the ER. A signal consisting of a short, four amino residue, known as the ER retention signal, is required. The signal found at the C-terminal of a resident ER lumen protein, BiP, is Lys-Asp-Glu-Leu. This sequence does not anchor the protein to the ER membrane, instead it causes any molecules detected in the Golgi to be cycled back. Experiments reveal the potency of this small signal.
With the exception of mitochondria, chloroplasts, nuclei, and peroxisomes, all other organelles receive their proteins via the rER. Many proteins are modified by the Golgi apparatus prior to delivery. Additional signals on the protein must direct the delivery machinery to place it in the correct location. Certain proteins not only have signal peptides but also possess signal patches, e.g. lysosomal hydrolases, where the patch allows proper modification of the hydrolase by special phosphotransferases.
It is not unusual to observe C-terminal signal peptides.
All cells possess a constitutive secretory pathway, where proteins destined for export are secreted from the cell. These proteins pass through the ER-Golgi processing pathway where modifications may occur. If no further signals are detected on the protein it will be directed to the cells surface for secretion. Some proteins are stored prior to release; these do have additional signals, and utilise the regulated secretory pathway.
Proteins can end up as integral membrane components. These proteins are said to be transmembrane, and initially follow the same pathway as those which pass through to the ER lumen, except that they are retained in the ER membrane by the presence of stop-transfer signals. Stop-transfer signals are hydrophobic stretches of about 20 amino acid residues that adopt an alpha-helical conformation as they transverse the membrane. Membrane embedded proteins are anchored in the phospholipid bilayer. The membrane can be distributed to different locations in the cell including the plasma membrane and organelle membranes. These membranes have a "sideness" and cannot flip over, i.e. a protein with its N-terminal facing the ER lumen will never face the cytosol.
Single membrane spanning proteins generally have a hydrophobic region that anchors that sequence in the phospholipid bilayer via a alpha-helix configuration. Proteins of this type can become embedded in the membrane in three ways:- In the simplest class, the N-terminal region has a signal peptide that passes through the membrane and is cleaved upon exiting into the lumen of the ER. However, a hydrophobic region in the central part of the polypeptide acts as a stop-transfer sequence. This leaves the C-terminal tail protruding from the cytosolic face of the ER. In another class, the signal peptide is internal on the polypeptide chain and is not cleaved. It acts as a start-transfer signal, initiating protein translocation, but upon release from the SRP receptor, anchors the peptide in the membrane. The only difference between this class and the last is the orientation of the signal peptide. It is reversed and results in the C-terminal of the polypeptide being directed into the ER lumen and the N-terminal in the cytoplasm. Multiple membrane spanning proteins result from the polypeptide chain passing back and forth across the phospholipid bilayer. Normally, the first helical domain is an internal uncleaved signal-start sequence that commences the transfer of the protein. The second helical region acts as a stop-transfer signal and prevents further transfer of the polypeptide. The result is the insertion of a hairpin loop across the membrane. Interaction with the SRP and a SRP receptor are necessary. Subsequent helical domains alternate as either start-transfer signal sequences or stop-transfer sequences. Each pair introduces another hairpin loop. Interestingly, SRP and SRP receptors are not involved. There are many examples of proteins of this type. A large class are known as heptahelicals, because they represent peptides that transverse the membrane seven times. The opsin class of proteins, which combine with a chromophore to generate the light stimulated molecules in the photoreceptors of animals, are a classic example. In this example (human cone opsin) of the hypothetical two-dimensional representation of the human long-wavelength or middle-wavelength sensitive opsin molecule, each of the seven transmembrane helices forms a alpha-helix as it passes through the phospholipid membrane (yellow blocks). The first helix has the characteristics of a start-transfer signal peptide, while the second helix is also a start-transfer signal and the third helix is a stop-transfer signal. The remaining four helical domains would alternate between start-transfer and stop-transfer signals.
Single membrane spanning proteins generally have a hydrophobic region that anchors that sequence in the phospholipid bilayer via a alpha-helix configuration. Proteins of this type can become embedded in the membrane in three ways:-
Multiple membrane spanning proteins result from the polypeptide chain passing back and forth across the phospholipid bilayer. Normally, the first helical domain is an internal uncleaved signal-start sequence that commences the transfer of the protein. The second helical region acts as a stop-transfer signal and prevents further transfer of the polypeptide. The result is the insertion of a hairpin loop across the membrane. Interaction with the SRP and a SRP receptor are necessary. Subsequent helical domains alternate as either start-transfer signal sequences or stop-transfer sequences. Each pair introduces another hairpin loop. Interestingly, SRP and SRP receptors are not involved.
There are many examples of proteins of this type. A large class are known as heptahelicals, because they represent peptides that transverse the membrane seven times. The opsin class of proteins, which combine with a chromophore to generate the light stimulated molecules in the photoreceptors of animals, are a classic example. In this example (human cone opsin) of the hypothetical two-dimensional representation of the human long-wavelength or middle-wavelength sensitive opsin molecule, each of the seven transmembrane helices forms a alpha-helix as it passes through the phospholipid membrane (yellow blocks). The first helix has the characteristics of a start-transfer signal peptide, while the second helix is also a start-transfer signal and the third helix is a stop-transfer signal. The remaining four helical domains would alternate between start-transfer and stop-transfer signals.
There are many examples of proteins of this type. A large class are known as heptahelicals, because they represent peptides that transverse the membrane seven times. The opsin class of proteins, which combine with a chromophore to generate the light stimulated molecules in the photoreceptors of animals, are a classic example.
In this example (human cone opsin) of the hypothetical two-dimensional representation of the human long-wavelength or middle-wavelength sensitive opsin molecule, each of the seven transmembrane helices forms a alpha-helix as it passes through the phospholipid membrane (yellow blocks). The first helix has the characteristics of a start-transfer signal peptide, while the second helix is also a start-transfer signal and the third helix is a stop-transfer signal. The remaining four helical domains would alternate between start-transfer and stop-transfer signals.
Signal Recognition Particle (SRP)
Secretory and other non-cytoplasmic proteins are synthesised only in association with the ER, and not with any other kind of membrane. Therefore the ER membrane must have some kind of identifying feature. The SRP, which consists of six polypeptide chains and a 300-nucleotide RNA, binds to the ER signal peptide as soon as it emerges from the ribosome.
Here is a diagram.
The interaction of the ribosome/polypeptide/SRP complex with the membrane is mediated by SRP receptor, a 650-residue integral membrane protein which may bind to the ribosome as well as to SRP. This protein is exposed on the cytosolic surface of rER. Closer analysis reveals the presence of GTP binding domains.
A number of ancillary factors ensure that the sorting signals are properly interpreted and that all proteins are delivered to their destinations. Specialised enzymes are responsible for the removal of the signal peptide sequences from proteins; they are known as signal peptidases. These enzymes are activated once the signal peptide has directed the protein to the desired location. Most compartments of organelles possess specific versions that only cleave a particular class, but not others.
Signal Peptide Detection
An on-line search engine offered by the Center for Biological Sequence Analysis, The Technical University of Denmark allows the detection of signal peptides.