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DNA and protein synthesis: Protein trafficking in



Proteins are an essential part of life. They are found throughout the body in many different forms, for example as receptors, signalling molecules and enzymes. Proteins are synthesised in the cytoplasm of cells, but this is not always necessarily where they are functionally needed. Mechanisms of protein transport are essential to deliver them to where they are needed. This article will discuss the different places where proteins are used, and how they get there.


Protein Targeting Overview

Protein synthesis


Proteins are synthesised using amino acids, from information found encoded in the genome. DNA is copied to mRNA, which then travels out of the nucleus into the cytoplasm; the site of protein synthesis.


An mRNA sequence is decoded in sets of three nucleotides, called codons. Each codon reads for an amino acid. There are four different nucleotides, so this would mean that there were 64 (4 x 4 x 4) possible combinations. There are however only 20 amino acids. This means that either there are some combinations that are never used, or different codons can code for the same amino acids. It is the latter theory that is correct, some amino acids are coded for by more than one codon.


A protein is not coded for directly from mRNA. Another type of RNA, transfer RNAs (tRNAS) act as adaptor proteins, to bring the amino acids together. tRNAs recognise the codon on one surface, and attach an amino acid on the other. A tRNA molecule will bind to the appropriate place on the mRNA with an amino acid attached. This amino acid will form a peptide bond with the next amino acid, also brought by a tRNA. This continues and a polypeptide chain forms.


It is not any use as a string of amino acids however. This polypeptide chain must fold up into its 3D conformation, bind any small molecule cofactors required for its activity, be appropriately modified by protein kinases or other protein modifying enzymes, and assemble correctly with other protein subunits with which it functions before it is functionally useful to the cell, and therefore the organism.



Protein targeting


Protein targeting, or protein trafficking, is the moving of proteins from their site of synthesis to the place where they are needed. This could be mitochondrial proteins encoded by nuclear DNA, receptor proteins being inserted into plasma membrane or signalling molecules being secreted from the cell, as well as many other examples.


Most proteins are synthesised in the cytoplasm of cells, where the ribosomes are located, but this is not always their place of function. There are two basic targeting pathways for proteins in eukaryotic cells: Post-translational and co-translational. The signals involved are called sorting signals. These are usually short sequences of amino acids, found either at the N-terminus of the protein, or integrated into its structure. They bind to specific receptors; either on the target organelle surface or on intermediate carrier proteins.


Post-translational targeting: e.g. proteins targeted for nucleus, mitochondria, chloroplasts. The signal is recognised after transcription is complete.


Co-translational targeting (secretory pathway): e.g. ER, Golgi, lysosomes, plasma membrane and secreted proteins. When the signal sequence is transcribed, the signal recognition particle (SRP) pauses transcription and redirects the mRNA, ribosome and partially translated peptide to a membrane. The SRP then docks into a receptor on a membrane surface and the protein is threaded through the membrane as it is translated.



Sorting signals


Each sorting signal (or signal peptide) represents a destination in the cell. For example, those proteins which are destined for the endoplasmic reticulum have a KDEL sequence. These amino acid sequences direct proteins synthesised in the cytoplasm to target organelles including the plasma membrane, nucleus, mitochondria and chloroplasts. A summary of the more common sequences is shown in the table below. Structural features of the protein are also thought to have a role in targeting, for example the phospholipid bilayer membranes of different organelles are different thicknesses. It is thought that proteins with shorter transmembrane domains tend to be retained in the endocytic pathway more than proteins with longer transmembrane domains which can span the thicker plasma membrane.



Protein targeting via the secretory pathway


The endoplasmic reticulum can bind ribosomes. A protein can be inserted into or through the ER membrane as it is being translated (co-translational). This is initiated with a signal sequence at the N-terminus of the emerging peptide chain. The signal sequence binds to an SRP (signal recognition particle), and translation is paused. The ribosome-complex then binds to the ER via a receptor and the signal sequence crosses the ER membrane. Translation then continues, with the peptide chain being pulled into the ER lumen. Whilst in the ER many proteins start to undergo glycoslation. The majority are packaged into vesicles, and enter the cis-phase of the Golgi- where they are further packaged and processed.


From the ER, proteins are dispatched to the Golgi and on to a range of locations. They may be packaged into vesicles for secretion or fused into the membranes of vesicles which then merge with the plasma membrane. Post-translational modification in the Golgi, such as the addition of mannose-6-phosphate may target proteins to lysosomes. Resident ER proteins contain a KDEL sequence which direct them back to the ER in COPI vesicles if they are transported to the Golgi.



ER import with SRP and SRPR

Mitochondrial localization


Mitochondria carry their own DNA and some mitochondrial proteins are produced on-site without needing trafficking. Most, however, are encoded by genomic DNA and need to be imported. These proteins are recognised by signal sequences which are typically amphipathic alpha helices.


As the precursor mitochondrial protein is produced from cytosolic ribosomes, the localization signal is recognised by chaperone proteins such as Hsc70. Hsc70 uses energy from ATP to keep the protein in an unfolded shape to allow it to be "threaded" though protein complexes into the mitochondria. The chaperone brings the protein to a Tom20/Tom22 receptor in the outer mitochondrial membrane. It is then transported through a transport channel complex including Tom40 across the outer membrane.


The protein is then transported across the inner mitochondrial membrane by a channel made of Tim proteins. (Tim 44, 23 and 17). This can happen at the same time as translocation through the TOM complexes because there are "contact" points in the mitochondria where the inner and outer membranes are very close together. This process also requires energy in the form of the proton gradient between the intermembrane space and the matrix which is created during respiration.


The Tim44 subunit has another chaperone attached called matrix Hsc70 which uses energy from ATP to continue to keep the protein from folding prematurely. Once translocation is complete this chaperone dissociates and proteases in the mitochondrial matrix cleave off the signal peptide. The peptide is then able (sometimes with the aid of other chaperone proteins such as Hsc60) to fold into the active form.



Nuclear localization


Some proteins need to be transported into the nucleus, as this is their site of action. A nuclear localisation sequence (NLS) is a sequence of amino acids which 'tags' the protein for import into the nucleus, by nuclear transport. The sequence is usually made up of positively charged lysines or arginines which are exposed on the cell surface. Different proteins may have the same NLS.


NLS's can be split in to two catorgories: Classical and non-classical. Classical NLS's can be divided further classified as either monopartite or bipartite.


Mechanism of Nuclear Import:

The nucleus is surrounded by envelope, which is made up of an inner and an outer membrane. The membrane consists of nuclear pores, which are the gateway for proteins. The nuclear pores are large protein complexes which cross the two membranes.


A protein which has been translated with a NLS sequence will bind strongly to importin (also called karyopherin), which is a protein that moves other protein molecules into the nucleus. Importin has two subunits, alpha and beta. Importin-beta can bind on its own, or alternatively can form heterodimers with importain-alpha (importin-alpha acts as an adaptor protein, binding to the NLS of the protein). Once the protein has been transported through the pore Ran-GTPase binds, and the importin complex dissociates. The protein is then released, and the importin/Ran-GTPase complex moves back out of the nucleus through the nuclear pore.



Targeted protein degradation


In order to remain working effieciently a cell must have mechanisms in place to remove proteins that are no longer needed. This may include: damaged proteins, incorrectly synthesised proteins, or proteins which are only needed at specific times, for example during the cell cycle. The degraded proteins amino acids are then recycled by the cell, to make new proteins. I am going to talk about two different mechanisms for protein degradation: via lysosome and via ubiqutin labelling.



Lysosomes are vesicles that contain enzymes needed for the break down and digestion of proteins. These enzymes need to be confined to a vesicle, as they could cause damage to the inside of the cell. Lysosomes can bind to endocytosed vesicles, and digest their contents, or be released via exocytosis in to the extracellular space.


Ubiquitin Labelling:

Ubiqutin is a small protein, which can be attached to other proteins using an activating enzyme. These ubiquitin tagged proteins are now recognised by proteases which can be found in the cytosol, and degrade the tagged protein.




1. Alberts, B. et al (2008) Molecular Biology of the Cell 5th Edition. Garland Science Publishing

2.  Campbell, N. and Reece, J. (2005) Biology 7th Edition. Pearson Education Inc.

3. Lodish, H. et al (2000) Molecular Cell Biology 4th Edition. W. H. Freeman Publishing

4. Fullekrug, J. and Nilsson, T. (1998) Protein sorting in the Golgi complex. Biochimica et Biophysica Acta. 1404: 77-84.


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