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(This page has links to sections of Mark Dalton's Introduction to Cell Biology; the Cell Biology Notes at MIT will also be of use.)
Some proteins function in the cytoplasm of cells, while others are an integral component of membranes. Others are directed towards particular organelles, while a number of types are secreted (exported from the cell). Most proteins which are not cytoplasmic enter the lumen of the Endoplasmic Reticulum (E.R.) as they are synthesized.
The Endoplasmic Reticulum (E.R.) is an extensive network of flattened, membrane-bound sacs generally arranged in layers in the cell. Some E.R. has ribosomes associated with the outer (cytosolic) side of its membrane, and is called rough endoplasmic reticulum due to its studded appearance.
Refer to the Mark Dalton's section on the endoplasmic reticulum, and the section on Structure and Function of Organelles (including a diagram of the endoplasmic reticulum) in the Cell Biology Notes at MIT.
The polypeptide chains synthesized by the ribosomes bound to the rough E.R. enter the endoplasmic reticulum lumen. There is no difference between the ribosomes associated with E.R. and those which are free in the cytosol:
Therefore whether or not a ribosome binds to the E.R. is dictated by the type of protein which it is synthesizing.
The ribosome associates with the E.R. membrane by means of a protrusion of the large (60S) subunit into the lipid bilayer.
Proteins which enter the E.R. during synthesis, and which are therefore synthesized by ribosomes on the rough E.R., have a signal peptide of 16 to 30 residues at their N-terminus. This sequence is subsequently cleaved off (by signal peptidase).
The signal peptides of different proteins are not homologous, but they generally include several (4 to 12) hydrophobic residues, which are found to be essential for the polypeptide to cross the E.R. membrane. There is also a basic residue a few residues before the hydrophobic sequence.
Some examples of signal peptides (continuous hydrophobic sequences are in bold):
Preproalbumin M K W V T F L L L L F I S G S A F S Pre-IgG light chain M D M R A P A Q I F G F L L L L F P G T R C Prelysozyme M R S L L I L V L C F L P L A A L G Preprolactin M N S Q V S A R K A G T L L L L M M S N L Prepenicillinase M S I Q H F R V A L I P F F A F C L P V F A Prevesicular stomatitis M K C L L Y L A F L F I H V N C virus glycoprotein Prelipoprotein M K A T K L V L G A V I L G S T L L A G
Genetic engineering techniques can add a signal peptide to the N-terminus of cytoplasmic proteins, such as globin,which naturally have no such sequences; this results in the engineered protein entering the E.R. and having the signal sequence cleaved off.
In cell-free systems containing ribosomes but no E.R. membrane component, the mRNA of secretory protein sequences is translated to give the polypeptide complete with signal sequence, which is not cleaved. If membrane vesicles are then added to the system, the protein still does not enter them, and the signal peptide remains attached (in the majority of proteins; see below).
The transport of most proteins through the membrane is therefore cotranslational. However, membrane vesicles need not be present at the beginning of translation. In the system described above, addition of membrane before the approximately the first 70 residues have been polymerized is sufficient for normal transport and signal cleavage to occur.
There are exceptions to the cotranslational behaviour described above. Mitochondria and chloroplasts are organelles which contain very small amounts of DNA. Most of their proteins are however encoded by the nuclear genome, and are synthesized by free cytoplasmic ribsomes. The resulting polypeptides have N-terminal sequences which are removed after the protein has crossed the organelle membrane. Such sequences are clearly of a different nature to those which associate their translating ribosomes with the E.R. However, some yeast secretory proteins have been found to be capable of passing through the E.R. after translation is complete, but the process requires ATP.
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.
Secretory and other non-cytoplasmic proteins are synthesized only in association with the E.R., and not with any other kind of membrane. Therefore the E.R. membrane must have some kind of identifying feature. The signal recognition particle (SRP) consists of six polypeptide chains and a 300-nucleotide RNA. This was identified by stripping E.R. membrane of certain membrane proteins, without which the membrane cannot accommodate the secretory polypeptide chains.
In the absence of E.R. membrane vesicles, but with SRP present, the synthesis of secretory proteins is halted when the polypeptide is approximately 70 amino acids long. Addition of membrane vesicles allows translation to resume. 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.
Here is a diagram.
Prokaryotic cells have no organelles such as E.R., but they do have ribosomes bound to the plasma membrane which synthesize secreted proteins, such as maltodextrin-binding protein which is secreted into the space between the plasma membrane and the cell wall (the periplasmic space) in gram negative bacteria. Such secreted proteins have similar N-terminal peptide sequences to eukaryotic secreted proteins, which are cleaved following secretion. Genetically engineered varieties of maltose-binding protein, in which only a single hydrophobic residue of the signal peptide has been replaced by a charged one, are not secreted, but remain in the cytoplasm complete with attached signal sequence.
Other examples, all with a similar type of fold, include
So far only proteins synthesized by the ribosomes of the rough E.R. have been considered. However, most of the proteins of the mitochondria and chloroplasts are synthesized in the in the cytoplasm by free ribosomes, and are subsequently imported into these organelles, which requires crossing their membranes. Some mitochondrial proteins are destined for the matrix, others for the intermembrane space, and others, such as those of cytochrome c oxidase, for the inner membrane. Some cytoplasmically-synthesized proteins also end up in peroxisomes.
Most of the mitochondrial proteins produced in the cytoplasm are synthesized as precursors which have N-terminal signal sequences of 20-60 residues (exceptions include cytochrome c, which is synthesized in the cytoplasm as the apo- form; after it has entered the mitochondrion, addition of haem is thought to prevent its transport in the opposite direction). The uptake of these precursors requires ATP. The conformation of the soluble precursors would be expected to be different to that of the mature protein, particularly in the cases of integral membrane proteins.
Some yeast mitochondrial proteins, such as F1-ATPase, are thought to bind in their precursor form to a receptor on the outer mitochondrial membrane. After the energy-requiring insertion through the membranes into the matrix, the signal sequence is cleaved (by a metalloprotease). During or after this cleavage, folding into the mature conformation occurs.
More complicated events accompany the insertion of cytochrome b2 (which occurs in the intermembrane space of the mitochondria) and cytochrome c1 (which is localized to the outer face of the inner membrane). The precursors of both of these proteins are partially inserted through the membrane so that a portion is in the matrix, where a section of the signal sequence is cleaved. The remaining signal residues are then removed and the haem prosthetic group is added.
Last updated 27th Nov '96