General Ribosomes Nuclear Signal Peptides Mitochondrial Signal Peptides ER Protein Trafficking Signal Patches
General
Ribosomes
Nuclear Signal Peptides
Mitochondrial Signal Peptides
ER Protein Trafficking
Signal Patches
Once signal peptides are identified their functionality can be experimentally tested by engineering them into other proteins by utilising the tools of molecular biology. In this way specific properties of novel signal peptides can be understood. For example, transferring the amino-terminal endoplasmic reticulum signal peptide on to the beginning of a cytosolic protein has resulted in that protein being directed to the reticulum. These types of experiments were key in determining the functional properties of signal peptides.
Site-directed mutagenesis (a means of changing specific amino acids) has been used to characterise the effects of altering the amino acids that comprise the signal sequence, and thus determine structural features
By simply labelling cellular free ribosomes with a suitable tag that can be tracked, it was clearly proved that any of the cells complement of ribosomes may engage in membrane-bound or membrane-free protein synthesis, with frequent switching between the two locations.
Gold Beads
Gold beads which are coated with a nuclear protein , but injected into the cytoplasm, are seen to migrate to the nuclear pores before appearing in the nucleus itself. Beads coated with a non-nuclear protein remain in the cytosol.
SV40 T-antigen
Experimentation involving mutation of the SV40 T-antigen have revealed that seven amino acids at the N-terminal end of the protein constitute the signal recognition peptide. Mutation of even one of these residues results in failure of the protein to enter the nucleus. Indeed transferring these seven amino acids to a cytoplasmic protein results in it migrating into the nucleus.
Nucleoplasmin
The frog nuclear protein, nucleoplasmin, exists as a pentamer (five molecules attached together) with each the C-terminal tails possessing a nuclear targeting signal. Injection of this protein into the cytoplasm of a frog egg soon results in its accumulation within the nucleus. Experiments that remove the tails from all these proteins prohibits the pentameric core from entering the nucleus. However, if a single tail is left on just one arm of the pentamer, the protein is re-targeted to the nucleus once more.
Phosphorylation of Signal Peptides
Some proteins may not be required at the time of their synthesis. One known mechanism by which these proteins are prevented from entry into the nucleus is the phosphorylation of their signal peptides. Enzymes in the cytoplasm add phosphate groups to certain amino acid residues and inactivate the signal. When needed these groups are removed and the protein is delivered to the nucleus.
Inhibitory Cytosolic Proteins
The signal peptides of certain proteins are masked by binding of inhibitory factors, which stops nuclear importation. The hormone receptors are good examples. The glucocorticoid receptor of a non-stimulated cell in tightly bound by a hsp90 factor. Upon activation by binding of a steroid molecule, the receptor disengages from the hsp90 molecule and is targeted into the nucleus.
Pulse-chase experiments
Pulse-chase experiments (involving the introduction of a brief input of radioactively labelled amino acids) demonstrate that proteins bearing mitochondrial uptake-targeting signals will enter mitochondria in both in vivo and in vitro systems. The radioactivity is initially detected on the outside of the mitochondria, then appearing inside them. The imported protein is shorter than that on the outside, because of the removal of the targeting signal. Protein uptake is demonstrated by the addition of protease. Only those within the mitochondria are protected from digestion.
Matrix-targeting Sequence
Addition of a matrix-targeting sequence onto the N-terminal of a protein which normally resides in the cytosol, results in that protein appearing in the mitochondrial matrix.
Mutation of Second Targeting Sequence
Proteins destined to compartments of the mitochondria other than the matrix carry two targeting sequences. In experiments where the second targeting signal is inactivated via mutation, the protein predictably remains in the matrix.
Endoplasmic Reticulum Protein Trafficking
Cotranslational Insertion
A series of experiments demonstrated that most proteins that pass through the rER membrane do so via a cotranslational process, which entails the simultaneous translation by the ribosome and its passage through the ER membrane. No energy input is necessary. In cell free extracts that consist of appropriate factors, but lack ER and SRPs, then proteins are synthesised with their signal peptides still intact. Addition of isolated ER membrane when synthesis commences results in the protein being deposited in the lumen of the ER with the signal peptide cleaved off. Should the membrane be added after the first 70 or so amino acids have been polymerised then the protein remains in the cytosol still possessing its signal sequence.
Interestingly, the same series of experiments repeated in the presence of SRP produces a different outcome: in the absence of ER membrane, protein synthesis arrests following the first 70 amino acids. If membrane is then added the protein appears in the ER lumen lacking its signal peptide. SRP binds to the growing peptide and stops translation until special membrane receptors are encountered.
ER Retention Signal
BiP is resident in the ER lumen. A short ER retention signal has been shown to achieve this. Deletion of the signal via molecular biological techniques, causes the protein to be secreted from the cell. When the signal is added to a secreted protein, that protein remains in the lumen of the ER.
The non-contiguous nature of amino acids comprising a signal patch make it difficult to locate these signals on proteins. However, the ability to generate fusion proteins (using the tricks of molecular biology) has make it possible to characterise these regions. A strategy known as epitope tagging can often be employed. A fusion protein consisting of the entire protein under investigation and a short 8 to 20 amino acid peptide is made. The location of the protein in the cell can be followed by using a specific antibody that binds to the peptide. Thus by altering any of the amino acid residues in the fusion protein, the effect on its intracellular location can be determined.