- Signal Sequences
- Tanslocation of Secretory Proteins Across the ER
- The signal sequences of secretory proteins to ER
- The signal recognition particle
- SRP receptor
- Translocation into the ER lumen
- Insertion of proteins into the ER membrane
- GPI-anchored proteins
- Protein Modifications in the ER
- Protein glycosylation
- Disulphide bond formation
- The role of N-linked glycosylation in ER protein folding
- Protein Targeting to Mitochondria and Chloroplast
- Transport of proteins to mitochondria
- Transport of proteins to chloroplast
- Protein Targeting to the Nucleus
- Nuclear localization signals
- Transport of proteins into the nucleus
- Transport of proteins out of the nucleus
- Transport and Sorting of Proteins to the Golgi Apparatus
- Transport of Proteins to the Lysosomes
- Receptor-Mediated Endocytosis and Sorting of Internalized Proteins
- Inhibitors of Protein Synthesis
- Diphtheria toxin
There are 10,000 different kinds of proteins in a mammalian cell. Most of these proteins are synthesized by cytosolic ribosomes and they remain within the cytosol. However, many of the proteins produced in a cell are delivered either to a particular cell membrane or to the cell surface for secretion. For example, many hormone receptor proteins and transporter proteins must be delivered to the plasma membrane and some water-soluble enzymes such as RNA and DNA polymerases and histones must be targeted to the nucleus. All the proteins produced by a cell must reach their correct locations for the cell to function properly. The delivery of newly synthesized proteins from the cytosol to their proper cellular locations is referred to as ‘protein sorting, protein targeting or protein trafficking’.
There are two basic forms of targeting pathways (Figure 6.1):
- Post-translational targeting: It occurs soon after the synthesis of protein by translation at the ribosome. These proteins are targeted to
- chloroplasts and
- Co-translational targeting (secretory pathway): Proteins as they are translated are targeted to the endoplasmic reticulum (ER) and thereby enter the secretory pathway. These proteins are targeted to
- Golgi apparatus,
- plasma membrane and
- secreted proteins.
Thus, these proteins are targeted post-translationally.
Signal sequences are the sequences that help in targeting proteins to their proper cellular destinations. These sequences are present in the synthesized protein itself. They are about 20–50 amino acids in length.
These signal sequences or uptake-targeting sequences are bound by receptor proteins. These govern the specificity of targeting. After binding with the receptor, the protein chain is transferred to a translocation channel that allows the protein to pass through the membrane bilayer. The energy required for this unidirectional transfer of a protein into an organelle, without sliding back into the cytosol, is achieved by coupling translocation to ATP hydrolysis.
Some proteins are subsequently sorted further to reach a sub-compartment within the target organelle; this requires yet other signal sequences and other receptor proteins. Once translocation across the membrane is completed, specific proteases remove signal sequences from the mature protein.
The same secretory pathway is used by all eukaryotic cells for synthesizing and sorting secreted proteins and soluble luminal proteins in the ER, Golgi apparatus and lysosomes. These proteins are collectively referred to as ‘secretory proteins’.
Although all cells secrete a variety of proteins (e.g., extracellular matrix proteins), certain types of cells are specialized for the secretion of large amounts of specific proteins. For example, pancreatic acinar cells synthesize large quantities of digestive enzymes that are secreted into ductules that lead to the intestine.
The Signal Sequences of Secretory Proteins to ER
Soon after the synthesis of a secretory protein on free ribosomes in the cytosol starts, a 16–30-residue ER signal sequence in the nascent protein directs the ribosome to the ER membrane and initiates translocation of the growing polypeptide across the ER membrane (Figure 6.2). An ER signal sequence is located at the N-terminus of the protein and consequently is the first part of the protein to be synthesized.
The signal sequences of different secreted proteins contain one or more positively charged amino acids that are adjacent to a continuous stretch of 6–12 hydrophobic residues. The signal sequence of most secretory proteins is cleaved from the protein while it is still growing on the ribosome and thus is usually not present in the mature proteins that are found in cells.
The Signal Recognition Particle
Signal recognition particles (SRPs) are the key components in protein targeting. SRP is a cytosolic ribonucleoprotein particle that transiently binds simultaneously to the ER signal sequence in a nascent protein, to the large ribosomal unit and to the SRP receptor that are present on the membrane of the ER. Six discrete polypeptides and 300-nucleotide RNA compose the SRP. One of the SRP proteins P54 is chemically cross linked to the ER signal sequences. The hydrophobic region of P54 contains a cleft which interacts with the hydrophobic N-termini of nascent secretory proteins. This selectively targets them to the ER membrane. The SRP proteins P9 and P14 interact with the ribosome, while the SRP proteins P68 and P72 are required for protein translocation.
Figure 6.2 The SRP cycle-protein transport into the ER lumen
The SRP receptor is an integral membrane protein that is made up of two subunits α and β. Apart from mediating the interaction of nascent secretory protein with the ER membrane, the SRP receptor also permits the elongation and completion of the protein.
Thus, the SRP and SRP receptor function to bring ribosomes that are synthesizing secretory proteins to the ER membrane. The energy from GTP hydrolysis is used to release proteins lacking proper signal sequences from SRP and SRP receptor complex, thereby preventing their mis-targeting to the ER membrane.
The interaction of the SRP–nascent chain–ribosome complex with the SRP receptor is promoted when GTP is bound by both P54 subunit of SRP and the α-subunit of SRP receptor.
This is followed by the transfer of the nascent chain and ribosome to a site on the ER membrane where translocation can take place. Hydrolysis of the bound GTP takes place. After dissociating, SRP and its receptor release the bound GDP and recycle to the cytosol for initiating another round of interaction between ribosomes synthesizing nascent secretory proteins for their co-translational import to the ER.
Translocation into the ER Lumen
Co-translational translocation into ER
After the targeting of the ribosome-synthesizing secretory protein to the ER membrane, the ribosome and nascent chain are rapidly transferred to the ‘translocon’, a protein-lined channel within the membrane (Figure 6.3). The process of translation continues and the elongating polypeptide passes directly from the large ribosomal subunit into the central pore of the translocon. The 60S ribosomal subunit is aligned with the pore of the translocon. The growing chain is never exposed to the cytoplasm and does not fold until it reaches the ER lumen. To maintain the permeability barrier of the ER membrane, the translocon is regulated, so that it is open only when a ribosome–nascent chain complex is bound. Thus, the translocon is a gated channel. When the translocon first opens, a loop of the nascent chain, containing the signal sequence and approximately 30 adjacent amino acids can insert into the translocon pore. As the growing polypeptide chain enters the lumen of the ER, the signal sequence is cleaved by signal peptidase, which is a transmembrane ER protein associated with translocon. This protease recognizes a sequence on the C-terminal side of the hydrophobic core of the signal peptide and cleaves the chain specifically at this sequence once it has emerged into the ER lumen. The translocon remains open until translation is completed and the entire polypeptide chain has moved into the ER lumen.
Figure 6.3 Co-translational translocation into the ER lumen
Post-translational translocation into ER
In most eukaryotes, secretory proteins enter the ER by co-translational translocation, using the energy derived from translocation to pass through the membrane. In yeast, however, some secretory proteins enter the ER lumen after translation has been completed, that is post-translational translocation (Figure 6.4). In this case, the translocating protein pass through the same translocon used in co-translational translocation; however, the SRP and SRP receptor are not involved in this case. In such cases, the direct interaction between the translocon and the signal sequence is sufficient for targeting to the ER membrane. In addition, the driving force for unidirectional translocation is provided by an additional protein complex known as the Sec63 complex and a member of the Hsc70 family of molecular chaperones known as Bip. The tetrameric Sec63 is embedded in the ER membrane in the vicinity of the translocon, while Bip is localized to the ER lumen. Bip has a peptide-binding domain and an ATPase domain. Bip binds and stabilizes the unfolded protein.
Figure 6.4 Post-translational protein import in to the ER
Once the N-terminal segment of the protein enters the ER lumen, signal peptidase cleaves the signal sequence. Bip–ATP interaction with the luminal portion of Sec63 complex causes the hydrolysis of the bound ATP, producing a conformational change in Bip that promotes its binding to an exposed polypeptide chain. In the absence of Bip, an unfolded polypeptide slides back within the translocon channel and thus does not allow the nascent polypeptide to enter the ER lumen. The Bip–ADP molecules bound to the polypeptide chain acts as a ratchet, ultimately drawing the entire polypeptide into the ER within a few seconds. Following this, the Bip molecules spontaneously exchange their bound ADP for ATP, leading to the release of the polypeptide, which can then fold into its native conformation. The recycled Bip–ATP is then ready for another interaction with Sec63.
Insertion of Proteins into the ER Membrane
Integral proteins located in ER, Golgi apparatus, lysosomal membranes and plasma membrane, which are synthesized on the rough ER that remain embedded in the membrane as they move to their final destinations along the same pathway followed by soluble secretory proteins. During this transport, the orientation of a membrane protein is preserved, i.e., the same segments of the protein always face the cytosol, while other segments always face the ER lumen. These sequences are collectively known as ‘topogenic sequences’, which direct the insertion and orientation of various classes of integral proteins into the membrane.
There are two major categories of hydrophobic signals used in the insertion of membrane proteins. All of these are membrane crossing domains:
- Start-transfer sequences: These are of two types:
- N-terminal signal peptide sequence: A cluster of about eight hydrophobic amino acids at the N-terminal end of a protein. This sequence remains in the membrane and is cleaved off of the protein after transfer through the membrane.
- Internal start-transfer sequence: Similar to a signal sequence, but located internally (not at the N-terminal end of the protein). It also binds to the SRP and initiates transfer. Unlike the N-terminal signal sequence, it is not cleaved after transfer of the protein.
- Stop-transfer signal: This is also a sequence of about eight hydrophobic amino acid residues. It follows either an N-terminal signal sequence or a start-transfer sequence. The stop-transfer signal is a membrane crossing domain. It remains in the membrane. The peptide is not cleaved.
- Start-transfer signal initiates the transfer of the carboxyl terminal arm of the polypeptide chain. When a stop-transfer peptide enters the translocator, it discharges the protein laterally into the membrane.
The topology of membrane proteins refers to the number of times that its polypeptide chains spans the membrane and the orientation of these membrane-spanning segments within the membrane. The key elements of a protein that determine its topology are the membrane-spanning segments themselves which usually contain 20–25 hydrophobic amino acids. Each of such segment forms an α-helix that spans the membrane with the hydrophobic amino acid anchored to the hydrophobic interior of the phospholipids bilayer. Based on the topogenic sequence, these integral transmembrane proteins are classified as ‘type I proteins’, when two sequences are involved in targeting and orienting them in the ER membrane, whereas ‘type II’ and ‘type III’ proteins contain a single, internal topogenic sequence.
Type I transmembrane protein insertion into the ER membrane
The signal sequence of all type I transmembrane proteins are located near their N-terminal. These proteins also have an internal hydrophobic sequence that becomes the transmembrane α-helix (transmembrane domain).
Like secretory protein, the N-terminal signal sequence on a nascent type I protein, initiates the co-translational translocation of the protein that is mediated through the combined action of the SRP and SRP receptor. Once the N-terminus of the growing polypeptide enters the lumen of the ER, the signal sequence is cleaved, and the growing chain continues to be extruded across the ER membrane.
A sequence of 22 hydrophobic amino acids in the middle of a type I protein stops the transfer of nascent chain through the translocon. This internal hydrophobic sequence can move laterally between the protein subunits that form the wall of the translocon. They get anchored in the phospholipid bilayer of the membrane, where it remains. This sequence that is responsible for the transmembrane anchoring of the protein is called a ‘stop-transfer anchor sequence’.
Once translocation is interrupted, translation continues at the ribosome, which is still anchored to the now unoccupied and closed translocon. As the C-terminus of the protein is synthesized, it loops out on the cytosolic side of the membrane. When translation is completed, the ribosome is released from the translocon and the C-terminus of the newly synthesized type I protein remains in the cytosol. That is the N-terminus region of the protein remains in the ER lumen, the hydrophobic region as the transmembrane domain and the C-terminal region as the cytosolic domain (Figure 6.5(a)).
Type II and type III transmembrane proteins insertion into the ER membrane
Unlike type I proteins, type II and type III proteins do not possess an N-terminal ER signal sequence. Instead they possess a single internal hydrophobic ‘signal-anchor sequence’ that functions both as an ER signal sequence and membrane anchor sequence. Based on the orientation of their respective signal anchor sequences within the translocon, type II and type III proteins have opposite orientation in the membrane.
In the case of type II proteins, after the internal signal anchor sequence is synthesized on a cytosolic ribosome, it is bound by an SRP. This directs the ribosome–nascent chain complex to the ER membrane. This is similar to targeting of soluble secretory proteins except that the hydrophobic signal sequence is not located at the N-terminus and is not subsequently cleaved. In the translocon, the N-terminal portion of the synthesizing protein is oriented towards the cytosol. As the chain is elongated and extruded into the lumen, the internal signal anchor moves laterally out the translocon. This hydrophobic sequence then anchors the polypeptide chain in the phospholipids bilayer.
Once protein synthesis is completed, the C-terminus of the polypeptide is released into the lumen and the ribosomal subunits are released into the cytosol. Thus, in this case, the N-terminus of the protein is oriented towards the cytosol and the C-terminus towards the ER lumen (Figure 6.5(b)).
In the case of type III proteins, the signal anchor sequence is located near the N-terminus, inserts the nascent chain into the ER membrane with its N-terminus facing the lumen, just the opposite of type II proteins. The signal sequence of type III proteins also prevent further extrusion of the nascent chain into the ER lumen, functioning as stop-transfer sequence. Translational elongation of the C-terminus in the cytosol continues and the hydrophobic sequence moving laterally between the translocon subunits anchor the polypeptide in the ER membrane.
Figure 6.5 (a) Type I transmembrane protein insertion into the ER membrane (b) Type II transmembrane protein insertion into the ER membrane
Some cell surface proteins are anchored to the phospholipid bilayer not by a sequence of hydrophobic amino acids but by a covalently attached amphipathic molecule, glycophosphatidylinositol (GPI). These proteins are synthesized and anchored to the ER membrane exactly like type I transmembrane proteins, with a cleaved N-terminal sequence and internal stop-transfer anchor sequence, directing the process. However, a short sequence of amino acids in the luminal domain, adjacent to the membrane-spanning domain, is recognized by a transamidase located within the ER membrane. This enzyme cleaves off the stop-transfer anchor sequence and transfers the remainder of the protein to a preformed GPI anchor in the membrane (Figure 6.6).
Figure 6.6 Membrane anchoring of proteins by GPI
Membrane and soluble secretory proteins that are synthesized on the rough ER undergo four principal modifications before they reach their final destination.
- Addition and processing of carbohydrates (glycosylation) in the ER and Golgi apparatus.
- Formation of disulphide bonds in the ER.
- Proper folding of polypeptide chains and assembly of multi-subunit proteins in the ER.
- Specific proteolytic cleavages in the ER, Golgi apparatus and secretory vesicles.
One or more carbohydrate chains are added to vast majority of proteins that are synthesized on the rough ER; indeed glycosylation is the principal chemical modification to most of these proteins. Carbohydrate chains in glycoproteins may be attached to the hydroxyl group in serine and threonine residues or to the amide nitrogen of asparagine. These are referred to as ‘O-linked oligosaccharides’ and ‘N-linked oligosaccharides’, respectively. O-linked oligosaccharides, such as those found in collagen and glycophorin, often contain only one to four sugar residues. The more common N-linked oligosaccharides are larger and more complex, containing several branches in mammalian cells. All N-linked oligosaccharides synthesis starts in the rough ER. A preformed oligosaccharide precursor containing 14 residues is attached to the protein. The branched oligosaccharide contains three glucose (Glc), nine mannose (Man) and two N-acetylglucosamine (GlcNAc)2 (Figure 6.7). This branched carbohydrate structure is modified in the ER and Golgi compartments, but five of the 14 residues are conserved in the structure of all N-linked oligosaccharides on secretory and membrane proteins.
The transfer of the oligosaccharide branch to the asparagine residue of the protein is catalysed by a membrane-bound enzyme, an oligosaccharyl transferase, which has its active site exposed on the luminal side of the ER membrane; this explains why cytosolic proteins are not glycosylated in this way. The precursor oligosaccharide is held in the ER membrane by a special lipid molecule called dolichol, and it is transferred to the target asparagine in a single enzymatic step immediately after that amino acid emerges in the ER lumen during protein translocation.
The oligosaccharide is assembled sugar by sugar onto the carrier lipid dolichol (a polyisoprenoid). Dolichol is long and very hydrophobic: its 22 five-carbon units can span the thickness of a lipid bilayer more than three times, so that the attached oligosaccharide is firmly anchored in the membrane. The first sugar group is linked to dolichol by a pyrophosphate bridge. This high-energy bond activates the oligosaccharide for its transfer from the lipid to an asparagine side chain of a nascent polypeptide on the luminal side of the rough ER. The synthesis of the oligosaccharide starts on the cytosolic side of the ER membrane and continues on the luminal face after the (Man)5 (GlcNAc)2 lipid intermediate is flipped across the bilayer. All of the subsequent glycosyl transfer reactions on the luminal side of the ER involve transfers from dolichol-P-glucose and dolichol-P-mannose; these activated and lipid-linked monosaccharides are synthesized from dolichol phosphate and UDP-glucose or GDP-mannose (as appropriate) on the cytosolic side of the ER and are then thought to be flipped across the ER membrane (Figure 6.8).
Figure 6.7 N-linked proteIn glycosylation
‘Tunicamycin’, an antibiotic produced by Streptomyces sp., mimics UDP-N-acetylglucosamine and blocks the first step in the synthesis of the core oligosaccharide of glycoproteins on dolichol phosphate. Tunicamycin group of antibiotics are produced by S. lysosuperficens. They contain uracil, N-acetylglucosamine, an 11-carbon aminodialdose called tunicamine, and a fatty acyl side chain. The structure of the fatty acyl side chain varies in the different members within the family. Apart from the variation in length of the fatty acyl side chain, some homologues lack the isopropyl group at the end and/or a 3-unsaturation.
Disulphide Bond Formation
Both intra-molecular and inter-molecular disulphide bonds (-S-S-) help stabilize the tertiary and quaternary structures of many proteins. The efficient formation of disulphide bonds in the lumen of the ER depends on the enzyme protein disulphide isomerase (PDI) which is present in all eukaryotic cells. This enzyme is especially abundant in the ER of secretory cells in the organs such as liver and pancreas.
Figure 6.8 Protein glycosylation in the ER lumen
Figure 6.9 PDI-assisted disulphide bond formation
The disulphide bond in the active site of PDI can be readily transferred to a protein by two sequential thiol-disulphide transfer reactions. The reduced PDI generated by this reaction is returned to its oxidized form by the action of a protein called Ero1, which carries a disulphide bond that can be transferred to PDI (Figure 6.9).
The Role of N-linked Glycosylation in ER Protein Folding
New soluble and membrane proteins produced in the ER generally fold into their proper conformation within minutes after their synthesis. The rapid folding of these proteins is mediated by the action of several proteins present in the ER called ‘chaperones’ (Figure 6.10).
The chaperone Bip not only helps co-translational translocation but also thought to prevent segments of a nascent chain from misfolding or forming aggregates, thereby promoting the folding of the polypeptide into proper conformation. PDI also contribute to proper folding.
The ER membrane-bound chaperone protein, ‘lectins’ (carbohydrate-binding protein) ‘calnexin’, binds to incompletely folded proteins containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER. The removal of the terminal glucose by a glucosidase releases the protein from calnexin. A glucosyltransferase is the crucial enzyme that determines whether the protein is folded properly or not: if the protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER. The cycle repeats until the protein has folded completely. ‘Calreticulin’ functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57, collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER.
Other important protein folding catalyst in the ER lumen is ‘peptidyl prolyl isomerases’, a family of enzymes that accelerate rotation about peptidyl-prolyl bonds. Unfolded or misfolded proteins are often transported to the cytosol for degradation.
Figure 6.10 Protein folding in ER lumen
Besides being bound by two membranes, both mitochondria and chloroplasts also contain similar type of electrotransport proteins and use an F-class ATPase to synthesize ATP. The growth and division of mitochondria and chloroplasts are not coupled to nuclear division.
Proteins encoded by the mitochondrial and chloroplast DNA are synthesized on the ribosomes within the organelles and directed to the correct sub-compartment immediately after their synthesis. The majority of proteins in mitochondria and chloroplasts, however, are encoded by genes in the nucleus and are imported into the organelle after their synthesis in the cytosol. Proteins synthesized in the cytosol that are destined for the matrix of mitochondria or to the stroma of chloroplasts contain specific N-terminal uptake-targeting sequences that specify binding to receptor proteins on the organelle surface. This targeting sequence is cleaved once it reaches the matrix or stroma (Figure 6.11).
Transport of Proteins to Mitochondria
Proteins targeted from the cytosol to same mitochondrial destinations have targeting signal sequences that share common motif. Thus, the receptors that recognize these signals are able to bind a number of different but related sequences. One of the sequences for localizing proteins to the mitochondria is the ‘matrix-targeting sequences’. These sequences are located at the N-terminus and are about 20–25 amino acids in length. They are rich in hydrophobic amino acids, basic amino acids (arginine and lysine) and hydroxylated ones (serine and threonine). These sequences lack negatively charged amino acids (acidic amino acids) such as aspartate and glutamate.
Figure 6.11 Translocons of mitochondrion and chloroplast
Mitochondrial ‘matrix-targeting sequences’ assume an a-helical conformation in which positively charged amino acids predominate on one side and hydrophobic amino acids predominate on the other side; thus, these sequences are amphipathic.
Transport of proteins to matrix of mitochondria
Proteins targeted to the mitochondria, soon after their synthesis interact directly with the mitochondrial membrane. Only unfolded proteins can be imported into the mitochondria. Chaperone proteins such as Hsc70 keep newly synthesized protein in an unfolded state. The proteins targeted to mitochondrial matrix takes a three-step route:
- First to the outer mitochondrial membrane,
- Second to the inner mitochondrial membrane and
- Finally to the mitochondrial matrix.
Transport to the outer mitochondrial membrane
The mitochondrial import of an unfolded protein is initiated by the binding of a mitochondrial sequence to an import receptor in the outer mitochondrial membrane. The N-terminal matrix-targeting sequences are recognized by proteins called Tom20 and Tom22. These are proteins in the outer mitochondrial membrane which are involved in targeting; Tom for translocon of the outer membrane.
The import receptor subsequently transfers the proteins to an import channel in the outer membrane. This channel is mainly composed of the Tom40 proteins. This is known as ‘import pore’, because all mitochondrial proteins gain access to the interior compartments of the mitochondria through this channel. Tom40 forms largely passive channel. These are wide enough to accommodate an unfolded protein, through the outer mitochondrial membrane.
Transport to the inner mitochondrial membrane
Transfer through the outer membrane occurs simultaneously with the transfer through an inner membrane channel. The inner membrane channel is composed of proteins called Tim23 and Tim17 proteins (Tim for translocon of inner membrane) (Figure 6.12).
Figure 6.12 Protein targeting to mitochondrial inter membrane space
(1a) Protease cleavage of matrix targeting sequence.
(1b) Protease cleavage of matrix targeting sequence.
(2a) Transport of cleaved protein with intermembrane space targeting sequence through channel proteins.
(2b) Transmembrane insertion of the intermembrane space targeting sequence; cleavage and release of the protein into the intermembrane space.
(3a) Cleavage of the intermembrane space targeting sequence and release into intermembrane space.
Transport to the mitochondrial matrix
Translocation into the mitochondrial matrix occurs at ‘contact site’ where outer and inner membranes are in close proximity. Soon after the AMterminal matrix-targeting sequence enters the mitochondrial matrix, it is removed by the protease that is present within the matrix. The emerging protein is also bound by the protein that is present in the translocation channels in the inner mitochondrial membrane known as matrix Hsc70. This binding requires interaction with Tim44. This interaction stimulates ATP hydrolysis by matrix Hsc70 which powers the translocation of proteins into the matrix. Some imported proteins can fold into their final active conformation without further assistance. However, many proteins require the help of a chaperonin for their final folding (Figure 6.13).
Transport of proteins to inner mitochondrial membrane
Three separate pathways are known to target proteins to the inner mitochondrial membrane.
One pathway uses the same machinery that is used for targeting of matrix proteins. A cytochrome oxidase subunit called Cox Va is a typical protein transported by this pathway.
Figure 6.13 Protein targeting to mitochondrial matrix
(1a, 2) Matrix targeting sequence directing the protein to outer membrane.
(1b) Binding of the protein with chaperonin Hsc70.
(3a, 4, 5) Transport of the protein through translocation channel powered by the energy of hydrolysis of ATP.
(6, 7a) Release of the protein into the matrix.
(7b) Hsc70 assisted folding of the protein
The second pathway to the inner membrane involves the use of matrix-targeting sequence and an internal hydrophobic domains that are recognized by an inner membrane protein termed Oxa1. The proteins are translocated (at least a portion of the protein) into the matrix through the Tom20/Tom22 and Tim23/17 channels.
After the cleavage of the matrix-targeting sequence, the protein is inserted into the inner membrane. This process requires the interaction with Oxa1 and other inner membrane proteins. Oxa1 also participates in the inner membrane insertion of certain proteins (e.g., subunit II of cytochrome oxidase) that are encoded by mitochondrial DNA and synthesized in the matrix by mitochondrial ribosome.
The final pathway for insertion in the inner mitochondrial membrane is followed by multipass proteins that contain six membrane-spanning domains such as ADP/ATP antiporter. These proteins, which lack the usual N-terminal matrix-targeting sequence, contain multiple internal mitochondrial targeting sequence. After the internal sequences are recognized by Tom70, the imported protein pass through the outer membrane through the general import pore. The protein is then transferred to a second translocation complex in the inner membrane that is composed of Tim9 and Tim10, which reside in the inner membrane space. These act as chaperones to guide imported protein from the general import pore to the Tim22/54 complex which incorporates the multiple hydrophobic segments of the imported protein into the inner membrane.
Transport of proteins to inter membrane space
The space that is present between the outer and inner mitochondrial membranes is called the intermembrane space. Two pathways deliver cytosolic proteins to the intermembrane space.
One pathway involves the use of an N-terminal sequence (hydrophobic sequence) and an N-terminal matrix-targeting sequence. Both of these sequences are finally cleaved. The N-terminal sequence (hydrophobic sequence) blocks the complete translocation of the protein across the inner membrane. The hydrophobic sequence after embedding the protein then diffuses laterally away from Tim23/17 translocation channel.
A protease in the inner membrane cleaves the protein near the hydrophobic transmembrane segment thus releasing the mature protein in the soluble form into the intermembrane space.
Cytochrome C heme lyase, the enzyme responsible for the attachment of heme to cytochrome C, explains the second pathway for targeting to the intermembrane space. In this pathway, imported protein is delivered directly to the intermembrane space via the general import pore without the involvement of any inner membrane translocation factors. Since translocation through the Tom40 general import pore is not coupled to any energetically favourable process such as the hydrolysis of ATP or GTP, the unidirectional translocation through the outer membrane is unclear. One possibility is that cytochrome C heme lyase passively diffuse through the outer membrane and then is trapped within the intermembrane space by binding to another protein that is delivered to that location by one of the translocation mechanisms discussed previously.
Transport of proteins to outer mitochondrial membrane
A short matrix-targeting sequence at the N-terminus of mitochondrial porin (P70) followed by a long stretch of hydrophobic sequence is involved in this translocation. Long stretch of hydrophobic sequence functions as a stop-transfer sequence that prevents the transfer of the protein into the matrix and anchors it as an integral protein in the outer membrane.
Transport of Proteins to Chloroplast
The chloroplasts have three membranes: outer membrane, inner membrane and the thylakoid membrane; consequently, the chloroplasts have three membrane spaces, which are intermembranous space, the stroma and the thylakoid membrane space. Proteins need to go through each of these membranes and into their respective compartments.
Figure 6.14 Protein targeting in plant cell (a) Free ribosomes in cytosol (b) Membrane-bound ribosomes
Proteins synthesized on membrane-bound ribosomes are first translocated into the lumen of the ER and then transported to the Golgi apparatus. These proteins may subsequently be targeted to the plasma membrane or the tonoplast, secreted or sent to the vacuole (Figure 6.14).
Chloroplast proteins may be encoded by nuclear DNA or chloroplast DNA; the respective mRNAs are translated by ribosomes in the cytosol (80S ribosome) or in the chloroplast stroma (70S ribosome). Proteins made as a precursor polypeptide in the cytosol may be targeted to the outer membrane or may enter the chloroplast stroma, to the thylakoid membrane, thylakoid lumen or inner envelope membrane (Figure 6.15).
Mechanism of protein import into the chloroplast
The import of proteins from the cytosol to chloroplasts shares several characteristics with mitochondrial import. In both processes, the imported proteins are synthesized as cytosolic precursors containing N-terminal uptake-targeting sequences that direct each protein to its correct subcompartment and are subsequently cleaved. Protein import from the cytosol into the chloroplast stroma (equivalent to the mitochondrial matrix) occurs, as in mitochondria, at the points where the outer and inner organelle membranes are in close contact. Finally, the protein import into both organelles requires energy. Despite the similarities just noted, the mechanisms of chloroplast and mitochondrial protein import differ in various ways.
Figure 6.15 Protein targeting to chloroplast sub compartments
Protein transport to chloroplast stromal space
Among the proteins found in the chloroplast stroma are the enzymes of the Calvin cycle. The large (L) subunit of ribulose-1,5-bisphosphate carboxylase (RuBisCO) is encoded by chloroplast DNA and synthesized on chloroplast ribosomes in the stromal space. The small (S) subunit of RuBisCO and all the other Calvin-cycle enzymes are encoded by nuclear genes and transported to chloroplasts after their synthesis in the cytosol (Figure 6.16).
The S-subunit of RuBisCO is synthesized on free cytosolic polyribosomes in a precursor form that has an N-terminal stromal-import sequence of about 44 amino acids. It is maintained in an unfolded state by binding to cytosolic chaperones; they can import the S-subunit precursor after its synthesis. After the unfolded precursor enters the stromal space, it binds transiently to a stromal Hsc70 chaperone, and the N-terminal sequence is cleaved. In the reactions that are facilitated by Hsc60 chaperonins, eight S-subunits combine with the eight L-subunits to yield the active RuBisCO enzyme.
Figure 6.16 Protein targeting to stroma of chloroplast
The import process involves the use of three chloroplast outer-membrane proteins, namely:
- A receptor that binds the stromal-targeting sequence,
- A transport channel protein and
- Five inner-membrane proteins.
Proteins are imported in the unfolded state into the stroma. The import process requires ATP hydrolysis that is catalysed by stromal chaperones, which functions similarly to Hsc70 of the mitochondrial matrix. Unlike mitochondria, however, chloroplasts cannot generate an electrochemical gradient (proton-motive force) across their inner membrane. Thus, protein import into the chloroplast stroma appears to be powered solely by ATP hydrolysis.
Targeting to the thylakoids
The targeting of proteins to thylakoid membrane or to the lumen involves the following steps (Figure 6.17):
- Transport to both the outer and inner chloroplast membranes to enter the stroma,
- Transport through the stroma and
- Transported protein can either be inserted into the thylakoid membrane or cross that membrane and enter the thylakoid lumen.
Figure 6.17 Protein targeting to thylakoids
Proteins that are destined for the thylakoid lumen, such as plastocyanin, require the successive action of two targeting sequences:
- The first sequence targets the protein to the stroma.
- The second sequence targets the protein from the stroma to the thylakoid lumen.
Four separate thylakoid-import systems, each transporting a different set of proteins from the stroma into the thylakoid lumen, have been identified.
One of the import systems is similar to the ER import and this system functions even in the absence of a pH gradient across the thylakoid membrane.
The other system involves the use of the thylakoid-membrane protein Hef106 which assists in translocating folded proteins and their bound cofactors into the thylakoid lumen. The protein uptake is powered by the pH gradient normally maintained across the thylakoid membrane.
The nucleus is separated from the cytoplasm by two membranes which form the ‘nuclear envelope’. The nuclear membrane acts as barrier that prevent the free passage of molecules between the nucleus and the cytoplasm. The transport of macromolecules including mRNA, tRNAs and ribosomal subunits out of the nucleus and the transport of all nuclear proteins occur through the ‘nuclear pore’ (Figure 6.18). Numerous pores perforate the nuclear envelope in all eukaryotic cells. Each nuclear pore is formed from an elaborate structure termed the ‘nuclear pore complex’ (NPC). NPC is made up of multiple copies of some 50–100 different proteins called ‘nucleoporins’, which allow the regulated exchange of molecules between the nucleus and the cytoplasm. The selective traffic of proteins and RNAs through the NPCs not only establishes the internal composition of the nucleus, but also plays a critical role in regulating eukaryotic gene expression.
Figure 6.18 Nuclear transport and nuclear pore
The nuclear membranes act as a barrier that separates the contents of the nucleus from the cytoplasm. Like other cell membranes, the nuclear membranes are phospholipid bilayers, which are permeable only to small non-polar molecules. The inner and outer nuclear membranes are joined at NPCs. These act as channels through which small polar molecules and macromolecules are able to travel through the nuclear envelope.
Small molecules and some proteins with molecular mass less than approximately 50 kDA pass freely across the nuclear envelope in either direction: cytoplasm to nucleus or nucleus to cytoplasm. They travel through open aqueous channels that are estimated to have diameters of approximately 9 nm, in the NPC. Most proteins and RNAs cannot pass through these open channels. These macromolecules pass through the NPC by an active process in which appropriate proteins and RNAs are recognized and selectively transported in only one direction (nucleus to cytoplasm or cytoplasm to nucleus).
The nuclear pore channels in the NPC, in response to appropriate signals, can open to a diameter of more than 25 nm. This size is sufficient to accommodate large ribonucleoprotein complexes, such as ribosomal subunits. Through these regulated channels, the nuclear proteins are selectively imported from the cytoplasm to the nucleus while RNAs are exported from the nucleus to the cytoplasm.
The NPC consists of an assembly of eight spokes attached to rings on the cytoplasmic and nuclear sides of the nuclear envelope. The spoke-ring assembly surrounds a central channel containing the central transporter.
Nuclear Localization Signals
All proteins found in the nucleus are synthesized in the cytoplasm and imported into the nucleus through the NPC. Such proteins contain a ‘nuclear localization signal (NLS)’ that directs their selective transport into the nucleus (Figure 6.19). Most of these sequences, like that of T-antigen, are short stretches rich in basic amino acid residues (lysine and arginine). In many cases, however, the amino acids that form the NLS are close together but not immediately adjacent to each other. For example, the NLS of nucleoplasmin (a protein involved in chromatin assembly) consists of two parts: a Lys-Arg pair followed by four lysines located 10 amino acids farther downstream. Both the Lys–Arg and Lys–Lys–Lys–Lys sequences are required for nuclear targeting.
Figure 6.19 Nuclear localization signals
Transport of Proteins into the Nucleus
Protein import through the NPC can be divided into two steps, based on their requirement for energy (Figure 6.20). The first step does not require energy. In this step, proteins that contain NLS bind to the NPC but do not pass through the pore. The NLSs are recognized by a cytosolic receptor protein, and the receptor–substrate complex binds to the nuclear pore. ‘Karyopherins’ are a group of importin-β super-family proteins that are involved in transporting molecules through the pores of the nuclear envelope. Karyopherins may act as importins or exportins.
The receptor, called importin, consists of two subunits. One subunit (importin-α) binds to the basic amino acid-rich NLSs of proteins such as T-antigen and nucleoplasmin. The second subunit (importin-β) binds to the cytoplasmic filaments of the NPC, bringing the target protein to the nuclear pore. Other types of NLSs, such as those of ribosomal proteins, are recognized by distinct receptors which are related to importin-β and function similarly to importin-β during the transport of their target proteins into the nucleus.
The second step in nuclear import, translocation through the NPC, is an energy-dependent process that requires GTP hydrolysis. A GTP-binding protein called ‘Ran’, which is related to the Ras proteins, is involved in the process.
Enzymes that stimulate GTP binding to Ran are localized to the nuclear side of the nuclear envelope. Enzymes that stimulate GTP hydrolysis are localized to the cytoplasmic side. As the result, there is a gradient of Ran/GTP across the nuclear envelope, with a high concentration of Ran/GTP in the nucleus and a high concentration of Ran/GDP in the cytoplasm.
This gradient of Ran/GTP determines the directionality of nuclear transport. GTP hydrolysis by Ran provides the energy required for nuclear import. Importin-β forms a complex with importin-α and its associated target protein on the cytoplasmic side of the NPC, in the presence of a high concentration of Ran/GDP. This complex is then transported through the nuclear pore to the nucleus, where a high concentration of Ran/GTP is present. At the nuclear side of the pore, Ran/GTP binds to Importin-β, displacing importin-α and the target protein. As a result, the target protein is released within the nucleus. The Ran/GTP-Importin-β complex is then exported to the cytosol, where the bound GTP is hydrolysed to GDP, releasing Importin-β to participate in another cycle of nuclear import.
Figure 6.20 Nuclear import of proteins through the nuclear pore
The nuclear import of transcription factors is regulated directly by their phosphorylation. For example, the yeast transcription factor SWI5 is imported into the nucleus only at a specific stage of the cell cycle. Otherwise, SWI5 is retained in the cytoplasm as a result of phosphorylation at serine residues adjacent to its NLS, preventing nuclear import. Regulated dephosphorylation of these sites activates SWI5 at the appropriate stage of the cell cycle by permitting its translocation to the nucleus.
Transport of Proteins Out of the Nucleus
RNAs are transported across the nuclear envelope as RNA–protein complexes. These proteins are recognized by exportins and transported from the nucleus to the cytoplasm. Pre-mRNAs and mRNAs are associated with a set of at least 20 proteins (forming heterogeneous nuclear ribonucleoproteins (hnRNPs)) throughout their processing in the nucleus and eventually transport to the cytoplasm. At least two of these hnRNP proteins contain nuclear export signals and are thought to function as the carriers of mRNAs during their export to the cytoplasm; ribosomal RNAs are assembled with ribosomal proteins in the nucleolus and intact ribosomal subunits are then transported to the cytoplasm. Their export from the nucleus appears to be mediated by nuclear export signals present on ribosomal proteins. The tRNA must mature inside the nucleus before export. tRNAs are aminoacylated and only tRNAs charged with an amino acid are exported efficiently. Export occurs when the tRNA is carried through the nuclear pore by a complex of exportin-t and Ran/GTP. Exportin-t binds to tRNA which in turn complexes with Ran/GTP. This complex diffuse through the channel filled with FG proteins, which actually generate hydrophobic environment.
In contrast to mRNAs, tRNAs and rRNAs, which function in the cytoplasm, the snRNAs function within the nucleus as components of the RNA processing machinery. These RNAs are initially transported from the nucleus to the cytoplasm, where they associate with proteins to form functional snRNPs and then return to the nucleus. Proteins that bind to the 5′ caps of snRNAs appear to be involved in the export of the snRNAs to the cytoplasm, whereas sequences present on the snRNP proteins are responsible for the transport of snRNPs from the cytoplasm to the nucleus.
Transport and Sorting of Proteins to the Golgi Apparatus
The Golgi apparatus is sometimes referred to as ‘the post office of the cell’, as it processes proteins made by the ER and sends them out to their various destinations in the cell. Proteins enter the Golgi apparatus on the side that is facing the ER (cis side) and exit on the opposite side of the stack, which is facing the plasma membrane of the cell (trans side) (Figure 6.21).
Proteins make their way through the Golgi apparatus stack of intervening cisternae and along the way become modified. They are then packaged for transport to various locations within the cell. The Golgi apparatus cisternae vary in number, shape and organization in different cell types. There are three major cisternae (cis, medial and trans). Sometimes additional regions are added to either side, which are called the cis-Golgi network (CGN) and the trans-Golgi network (TGN). These networks have a more variable structure, including some cisterna-like regions and some vesiculated regions.
Different protein modification enzymes are present in each cisterna or the region of the Golgi apparatus. The Golgi enzymes catalyse the addition or removal of sugars from cargo proteins (glycosylation), the addition of sulphate groups (sulphation) and the addition of phosphate groups (phosphorylation). The enzymes sequentially add the appropriate modifications to the proteins.
Some Golgi-mediated modifications act as signals to direct the proteins to their final destinations that are present within cells; for example, the lysosome and the plasma membrane. Defects in various aspects of Golgi function can result in congenital glycosylation disorders, some forms of muscular dystrophy and may contribute to diabetes, cancer and cystic fibrosis.
Figure 6.21 Transport of proteins by the Golgi vesicles
The Golgi apparatus is often found in close proximity to the ER in cells. Protein cargo moves from the ER to the Golgi apparatus, is modified vesicles to the Golgi apparatus, within the Golgi apparatus and is then sent to various destinations in the cell, including the lysosomes and the cell surface. Cargo proteins move between the Golgi cisternae by two possible explanations: the vesicular transport model and cisternal maturation model (Figure 6.22).
Figure 6.22 Two models of protein trafficking through the Golgi apparatus (a) The cisternal maturation model of protein movement through the Golgi apparatus. As a new cis cisterna is formed, it traverses the Golgi stack, changing as it matures by accumulating medial, then trans enzymes through vesicles that move from later to earlier cisternae (retrograde traffic). (b) The vesicular transport model, where each cisterna remains in one place with unchanging enzymes and the proteins move forward through the stack via vesicles that move from earlier to later cisternae (anterograde traffic)
Transport of Proteins to the Lysosomes
Proteins targeted to the lysosomes, for example enzymes such as hydrolases, upon arrival in the Golgi complex from the ER, their signal patch is recognized by a phosphotransferase that catalyses the phosphorylation of certain mannose residues in the enzymes’ oligosaccharides. The presence of one or more mannose-6-phosphate residues in their N-linked oligosaccharides is the structural signal that targets these proteins to lysosomes.
A receptor protein in the membrane of the Golgi complex recognizes this mannose-6-phosphate signal and binds the hydrolases so marked. Vesicles containing these receptor-hydrolase complexes bud from the trans side of the Golgi complex and make their way to sorting vesicles.
Inside the sorting vesicle, the receptor hydrolase complexes dissociate in a process facilitated by the lower pH within the sorting vesicles and by a phosphatase-catalysed removal of phosphate groups from the mannose-6-phosphate residues.
The receptor is then returned to the Golgi complex. Vesicles that are containing the hydrolases bud from the sorting vesicles and move to the lysosomes.
In cells that are treated with tunicamycin and hydrolases normally targeted for lysosomes do not reach their destination but are secreted instead, confirming that the N-linked oligosaccharide plays a key role in targeting these enzymes to lysosomes.
Some proteins such as low-density lipoprotein (LDL), the iron-carrying protein transferrin, peptide hormones and circulating proteins, which are destined to be degraded, are imported into certain cells from the surrounding medium. These proteins bind to receptors on the outer face of the plasma membrane. These receptors are concentrated in the invaginations of the membrane called coated pits, which are coated on their cytosolic side with a lattice made up of the protein called clathrin.
Clathrin forms closed polyhedral structures. Clathrin is a trimer of three light (L) chains and three heavy (H) chains. The (HL)3 clathrin unit is organized as a three-legged structure called a ‘triskelion’. Triskelions have a propensity to assemble into polyhedral lattices (Figure 6.23).
As more of the receptors become occupied with target proteins, the clathrin lattice grows until a complete membrane-bounded endocytic vesicle buds off the plasma membrane and moves into the cytoplasm.
The clathrin is quickly removed by uncoating enzymes and the vesicles fuse with endosomes. The pH of endosomes is lowered by the activity of V-type ATPases in their membranes. This creates an environment that facilitates the dissociation of receptors from their target proteins. Proteins and receptors then go their separate ways.
Receptor-mediated endocytosis (Figure 6.24) is exploited by some viruses to gain entry to cells. Influenza virus enters cells this way. HIV, the virus that causes AIDS, also binds to specific receptors on the cell surface and may gain entry by endocytosis. In humans, the receptor that binds HIV, known as CD4, is a glycoprotein found primarily on the surface of immune system cells called helper T-cells. CD4 is normally involved in the complex communication between cells of the immune system that is required to execute the immune response.
Figure 6.23 Clathrin
Figure 6.24 Clathrin-mediated enocytosis
First, they have been very useful scientifically in elucidating the biochemical mechanisms of protein synthesis.
Second, some of these inhibitors affect prokaryotic but not eukaryotic protein synthesis and thus are medically important antibiotics.
Streptomycin is an aminoglycoside antibiotic that affects the function of the prokaryotic 30S sub-unit. Low concentrations of streptomycin induce mRNA misreading. As the result, improper amino acids are incorporated into the polypeptide. Codons with pyrimidines in the first and second positions are particularly susceptible to streptomycin-induced misreading. These reading errors are not frame shift mistakes; therefore, totally aberrant proteins are not made at low streptomycin levels. Thus, susceptible cells are not killed, but their growth rate is severely decreased. At high concentrations of streptomycin, non-productive 70S ribosome–mRNA complexes accumulate, preventing the formation of active initiation complexes with new mRNA.
Table 6.1 Some of the protein synthesis inhibitors and their action
|Inhibitor||Mode of action||Organism inhibited|
|INHIBITORS OF INITIATION|
|Aurintricarboxylic acid||Inhibits initiation factor binding to the 30S subunit||Prokaryotes|
|Kasugamycin||Inhibits f-Met-tRNAf Met binding||Prokaryotes|
|Streptomycin||Inhibits formation of initiation complexes||Prokaryotes|
|INHIBITORS OF ELONGATION|
|Tetracycline||Inhibits aminoacyl-tRNA binding at A site||Prokaryotes|
|Streptomycin||Leads to codon misreading, insertion of improper amino acid||Prokaryotes|
|Sparsomycin||Peptidyl transferase inhibitor||Prokaryotes|
|Chloramphenicol||Blocks peptidyl transferase activity by binding to 50S subunit||Prokaryotes|
|Erythromycin||Blocks peptidyl transferase activity by binding to 50S subunit||Prokaryotes|
|Cycloheximide||Inhibits translocation of peptidyl-tRNA||Eukaryotes|
|Fusidic acid||Inhibits EF-G:GDP dissociation from ribosome||Both prokaryotes and eukaryotes|
|Thiostrepton||Inhibits ribosome-dependent EF-Tu and EF-G GTPase||Prokaryotes|
|Diphtheria toxin||ADP-ribosylates and inactivates eEF2||Eukaryotes|
|INHIBITORS THAT BRINGS ABOUT PREMATURE TERMINATION|
|Puromycin||Aminoacyl-tRNA analogue, acts as a peptidyl acceptor and aborts further peptide elongation||Both prokaryotes and eukaryotes|
|Ricin||Inactivates 28S rRNA||Eukaryotes|
Figure 6.25 Inhibitors of protein synthesis
Figure 6.26 ADP-Ribosylation of the diphthamide moiety of eukaryotic EF-2
Puromycin structurally resembles 3′-end of aminoacyl-tRNAs. Puromycin binds at the A site of both prokaryotic and eukaryotic ribosomes. The binding is not dependent on EF-Tu (or EF1). Puromycin acts as an acceptor of the peptidyl chain from peptidyl-tRNA
Diphtheria arises from infection by Corynebacterium diphtheriae, a bacterium carrying bacteriophage corynephage. Diphtheria toxin is a phage-encoded enzyme secreted by these bacteria. The toxin is capable of inactivating a number of GTP-dependent enzymes. Diphtheria toxin is an NAD+-dependent ADP-ribosylase. It covalently attaches to an ADP-ribosyl moiety derived from NAD+.
One target of diphtheria toxin is the eukaryotic translocation factor, EF2, which has a modified His residue known as diphthamide. Diphthamide is generated post-translationally on EF2; its biological function is unknown. (EF-G of prokaryotes lacks this unusual modification and is not susceptible to diphtheria toxin.) Diphtheria toxin specifically ADP-ribosylates an imidazole-N within the diphthamide moiety of EF2 (Figure 6.26).
ADP-ribosylated EF2 can still bind GTP but cannot function in protein synthesis. As diphtheria toxin is an enzyme and can act catalytically to modify many molecules of its target protein, just a few micrograms sufficient to cause death.
Diphtheria toxin catalyses the NAD+-dependent ADP-ribosylation of selected proteins like ADP-ribosylation of the diphthamide moiety of eukaryotic EF2. (Diphthamide=2-[3-carboxamido-3-(trimethylammonio)propyl]histidine.)
Ricin is an extremely toxic glycoprotein produced by the plant Ricinus communis (castor bean). The protein is disulphide-linked. It is a heterodimer of roughly equal 30-kDA subunits namely A and B.
The A-subunit (32 kDA) is an enzyme and is the toxic subunit; ricin gains entry to cells with the help of the B-subunit (33 kDA) which is a lectin. (Lectins form a class of proteins that bind to specific carbohydrate moieties commonly displayed by glycoproteins and glycolipids on cell surfaces.)
Endocytosis of bound ricin followed by disulphide reduction releases the A chain. The A chain enters the cytosol and catalytically inactivates eukaryotic large ribosomal subunits. A single molecule of ricin A chain in the cytosol can inactivate 50,000 ribosomes and can kill a eukaryotic cell.
Ricin A chain specifically attacks a single and highly conserved adenosine (an A at position 4,256) in the eukaryotic 28S rRNA. Ricin A chain has an N-glycosidase activity that removes the adenine base, leaving the rRNA sugar–phosphate backbone intact. The removal of this single base is sufficient to inactivate a 60S large subunit. The adenine in this highly conserved region of the 28S rRNA sequence is believed to be crucial to the functions of the 60S subunit that involve EF1 and EF2.
- The delivery of newly synthesized proteins to their proper cellular destinations is referred to as protein sorting or protein targeting.
- There are two basic forms of targeting pathways:
- Post-translational targeting: It occurs soon after the synthesis of protein by translation at the ribosome. Proteins targeting to nucleus, mitochondria, chloroplasts and peroxisomes are targeted post-translationally.
- Co-translational targeting (secretory pathway): Proteins as they are translated are targeted to the ER and thereby enter the secretory pathway. These proteins include proteins targeted to ER, Golgi apparatus, lysosomes and plasma membrane secreted proteins. Thus, these proteins are targeted co-translationally.
- The information to target a protein to a particular organelle destination is encoded within the sequence of 20–50 amino acids, generally known as ‘signal sequences’ or ‘uptake-targeting sequences’.
- SRP are the key components in protein targeting. SRP is a cytosolic ribonucleoprotein particle that transiently binds simultaneously to the ER signal sequence in a nascent protein, to the large ribosomal unit and to the SRP receptor present on the membrane of the ER.
- Proteins can be transported to the ER either post-translationally or co-translationally.
- Proteins destined for the mitochondrial matrix or chloroplast stroma have organelle-specific N-terminal uptake-targeting sequences that direct their entry into the organelle. After protein import, the targeting sequence is removed by proteases within the matrix or stroma.
- Protein import into both mitochondria and chloroplasts occurs only at the sites where the inner and outer organellar membranes are in close contact.There are two major categories of hydrophobic signals used in the insertion of membrane proteins. All of these are membrane-crossing domains: start-transfer sequences and stop-transfer signal.
- The topology of membrane proteins refers to the number of times that its polypeptide chains spans the membrane and the orientation of these membrane-spanning segments within the membrane.
- Membrane and soluble secretory proteins synthesized on the rough ER undergo four principal modifications before they reach their final destination:
- Addition and processing of carbohydrates (glycosylation) in the ER and Golgi apparatus.
- Formation of disulphide bonds in the ER.
- Proper folding of polypeptide chains and the assembly of multisubunit proteins in the ER.
- Specific proteolytic cleavages in the ER, Golgi apparatus and secretory vesicles.
- New soluble and membrane proteins produced in the ER generally fold into their proper conformation within minutes after their synthesis. The rapid folding of these proteins is mediated by the action of several proteins present in the ER called chaperones.
What is meant by protein targeting? What is the role of SRPs in protein targeting?
Explain the process of insertion of proteins into the ER membrane.
Briefly explain the process of protein targeting to mitochondria and chloroplast.
Explain the role of nuclear localization signals.
Enlist atleast 5 inhibitors of protein synthesis. Briefly explain about any two inhibitors.
Explain the protocol for transport of proteins out of the nucleus.
Enumerate the significance of protein sorting.
The signals that target the proteins to their respective organelles are called
- leader peptides
- sorting signals
- signal sequences
- all of the given options
The transport of proteins to Golgi and lysosomes is
- post translational
- co translational
The proteins glycosylated on Asparagine residues are called
- N-Linked glyco proteins
- O-Linked glycoproteins
- glycol proteins
- all of the given options
The monosaccharides used in protein glycosylation
- all of the given options
——— is called the post office of a cell
- golgi complex
- endoplasmic reticulum
Synthesis of all polypeptides encoded by nuclear genes begins in the
- golgi complex
——— helps in the proper folding of nascent proteins
- HSP 70
- all of the given options
Proteins that are connected with nuclear transport are
- all of the given options
The inhibitor that blocks the peptidyl transferase activity during translation
The inhibitor of translation which isanalogous to aminoacyl t RNA and inhibits elongation process
- fusidic acid
- Alberts, Bruce, Bray, Dennis, Lewis, Julian, Raff, Martin, Roberts, Keith and Watson, James D. 1994. Molecular Biology of the Cell, 3rd edition, New York: Garland Science.
- Becker, Wayne M., Kleinsmith, Lewis J., Hardin, Jeff and Bertoni, Gregory Paul. 2008. The World of the Cell, 7th edition: Pearson Education Inc.
- Bolender, Natalia, Sickmann, Albert, Richard Wagner, Chris Meisinger and Nikolaus Pfanner. 2008. ‘Multiple Pathways for Sorting Mitochondrial Precursor Proteins’, EMBO Reports, 9(1): 42–49.
- Cooper, Geoffrey M. 2000. The Cell: A Molecular Approach, 2nd edition, Sunderland, MA: Sinauer Associates.
- Lehninger, Albert L., Nelson, David L. and Cox, Michael M. 2004. Lehninger Principles of Biochemistry. W H Freeman & Co.
- Lodish, Harvey, Berk, Arnold, Zipursky, S. Lawrence, Matsudaira, Paul and Baltimore, David. 2006. Molecular Cell Biology, 4th edition, New York: W H Freeman & Co.
- Schnell, D.J. 1998. ‘Protein Targeting to the Thylakoid Membrane’ Annual Review of Plant Physiology and Plant Molecular Biology, 49: 97–126.