The Rer/Golgi System (Part Two)
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THE RER/GOLGI SYSTEM (PART TWO)
Textbook: pp 696-702 (N.B. You are responsible for understanding the mechanisms of transcription and translation as covered in first year.)
The synthesis of most proteins starts and is completed in the cytosol.
Proteins that are meant to remain in the cytosol lack a targetting sequence. Proteins that must be put into nucleus, mitochondria, chloroplasts, or peroxisomes all have specific targetting sequences. Remember that for import into these organelles the polypeptides may remain folded (import into nucleus or peroxisomes) or may have to be unfolded before import into the organelle). Import into these organelles is called post-translational.
The synthesis of proteins destined for the RER starts in the cytosol most of the synthesis is co-translational.
As I mentioned previously (Part One) proteins that are imported into the RER (either inserted into the RER membrane or the lumen) have various final destinations:
(1)Proteins may remain and function in RER membrane e.g. proteins of the translocon and the signal peptidase.
(2)Proteins may remain and function in the RER lumen e.g. various chaperones.
(3)Proteins may remain and function in the membranes of the Golgi bodies e.g. the transport system for UDP-glucose.
(4)Proteins may remain in the lumen of Golgi cisternae e.g. glycosyl-transferases.
(5)Proteins may be incorporated into “pre-lysosomes”.
(6)Proteins may be incorporated into secretory vesicles.
(7)Proteins may be incorporated into vesicles that are primarily for synthesis of the plasma membrane.
(8)Proteins may be incorporated into melanosomes.
(9)Proteins may be incorporated into the central vacuole of plant cells.
This diagram is from p. 697 of your textbook, and clearly does not include all the possible destinations of proteins into the RER. Polypeptides destined for import into the RER have a targetting sequence consisting of about 20 amino acidsat the N-terminal end of the polypeptide. Note that the N-terminal end of a polypeptide is the end of the polypeptide that is synthesized first. The targetting sequence for import into the RER has the specific name of the signal sequence. It was the first evidence of a targetting sequence for any organelle. The concept (the signal hypothesis) that protein import into organelles requires a specific stretch of amino acid sequence (the targetting sequence) was put forward by Gunter Blobel who received the Nobel Prize in 1999 for elucidating the mechanism of co-translational import into the RER hypothesis. He likened targetting sequnces to zip codes. As your textbook says, he received the Nobel Prize because “this hypothesis (the signal hypothesis) so profoundly influenced the field of cell biology …”.
For the mechanism of protein import into the RER see your textbook (page 699). You should look at that diagram as you read the points listed below!
Listed below are the required steps;
(1)Initiation of synthesis of the polypeptide in the cytosol. This limited synthesis is to allow the signal sequence to be exerted from the ribosome.
(2)Binding of the signal receptor particle (SRP). The SRP is found in all cells, including prokaryotes. It is found in prokaryotes because the secretion of proteins across the call membrane of prokaryotes is similar to import into the RER.
(3)Binding of the SRP to the signal sequence immediately halts translation.
(4)The SRP/ribosome/mRNA complexes diffuseto the outer surface of the RER.
(5)The SRP, while still bound to the signal sequence, binds to the SRP receptor. The SRP receptor is an integral membrane protein of the RER membrane and is a part of the protein complex called the translocon.
(6)The SRP binding to its receptor brings the ribosome in contact with the ribosome receptor. The ribosome receptor is also part of the translocon complex. Binding of the ribosome to the ribosome receptor causes the translocon pore to open. The translocon pore thus only opens when it is capped by a ribosome! This is very important, because otherwise important small solutes would leak from the RER lumen into the cytosol.
(7)The SRP isunbound from the SRP receptor and it diffuses away. The energy for the unbinding process is the hydrolysis of GTP, not ATP. Both the SRP receptor and the SRP itself bind GTP and have GTPase activity.
(8)Translation starts again and the growing polypeptide is pushed and pulled into the RER lumen.
(9)The signal sequence is cut off by the signal peptidase. The signal peptidase is also an integral membrane protein of the RER membrane and a member of the translocon complex. The cut-off signal sequence is degraded to its individual amino acids by soluble peptidases in the RER lumen. The amino acids then enter the cytosol via amino acid transport systems in the RER membrane.
(10)The polypeptide folds into its correct secondary and tertiary structures.
How do the integral membrane proteins become inserted into the RER membrane?
We have seen above how water-soluble proteins end up in the RER lumen? But what about all the important proteins that must also be imported “into” the ER but be threaded into the membrane instead of being located completely in the lumen? Such proteins would, of course, include the SRP receptor protein, translocon pore protein itself, the ribosome receptor, and the signal peptidase. These proteins will remain in the RER membrane where they function. But will include many membrane proteins that will go on to the Golgi bodies in transport vesicles.
This diagram is form p.702 of your textbook. Following are the steps:
(1)These integral membrane polypeptides start off with a signal sequence.
(2)The signal sequence is soon cut off by the signal peptidase.
(3)Most integral membrane proteins dissolve , and are thus anchored in the membrane, because they have internal hydrophobic sequences (about 22 amino acids long). These hydrophobic sequences exist in the membrane as α-helices with hydrophobic R-groups poking out from the “surface” of the α-helix.
(4)Elongation of the polypeptide continues until the hydrophobic “stop-transfer” sequence is reached. (The “stop-transfer” sequence is really just the hydrophobic region that is going to exist as a membrane-spanning α-helix in the finished protein. It must be about 22 amino long to be able to from the required α-helix.
(5)The hydrophobic sequence, now an in the from of an α-helix diffuses sideways out of the translocon through a side-opening in the translocon that exists for just this function! (Steps #1-#5 are shown in this diagram (p. 702) from your textbook. I also showed this diagram at the begiinning of this discussion!)
(6)There is actually other, really very similar way. In which this can happen. In this case, the “stop-transfer” sequence also serves as the signal sequence!
The diagram below (from Alberts et al. “Cell Biology” 2008) gives an idea of how the translocon allows the integral protein to move sideways out of it. It also shows how the translocon cap is moved out of the way during translation (when a ribosome is serving as the translocon cap). The gap in the side of the translocon also allows the cut-off signal sequence of all imported polypeptides to move out into the membrane so it can be digested.
The insertion of proteins having more than membrane-spanning α-helix in its sequence is more complex. It seems to each stop-transfer sequence serving as a new signal sequence, with a new interaction with a ribosome and SRP for insertion of each α-helix into the RER membrane. In other words, a more complex version of the mechanism shown for a so-called “single-pass” polypeptide shown in (6) above.
The role of Hsp70 in import of polypeptides into the RER
There is another protein (in addition to the ribosomal proteins, the proteins of the translocon and the SRP) that are necessary for import of the growing polypeptide through the pore of the pore protein of the translocon. These proteins are called heat shockproteins (Hsp), or increasingly these days, heat shock chaperones (Hsc). The one we will talk about here is a member of the Hsp70 family. The “70” refers to the MW of the protein i.e 70,000 Daltons. In other words, the members of the Hsp70 family are only medium-sized proteins. They have a binding site (which is also an ATPase active site) for ATP and a biding fro hydrophobic sequences in polypeptides.
The Hsp proteins are called chaperones because they “take care of otherproteins”. For example they:
(1)Bind to proteins when proteins are above their optimal temperature, to prevent them from folding improperly when the temperature is lowered. In other words they reduce the chances of the thermal denaturation of proteins when they are in a cell.
(2)They bind to the unfolded polypeptides (proteins) as they are imported into mitochondria and chloroplasts.
(3)They bind to polypeptides as they are exerted from the ribosome. The tertiary structure of water-soluble proteins is largely driven by the interaction of hydrophobic regions of the secondary structure. This hydrophobic interaction causes the hydrophobic regions to be “hidden away” from water. But as a polypeptide is being synthesized it is the case that the particular hydrophobic region that another hydrophobic region is supposed to interact with has not yet been made! So in the meantime it is essential that the hydrophobic region that has already been made be prevented from improperly interacting (i.e. “gluing”) to hydrophobic regions it is not supposed to interact with. These improper hydrophobic regions could even on other polypeptides being synthesized by nearby ribosomes of the polyribosome!
(4)“Pulling” nascent polypeptides across RER membrane and across the membranes of mitochondria and chloroplasts.
Strictly speaking, the Hsp70 molecules do not actually “pull” the elongating elongating polypeptide across the membrane. The polypeptide is being pushed into the pore by the fact it is belong elongated. But it can still wiggle up and down in the pore to some extent by thermal motion. So when it is on a “wiggle down” an Hsp70 grabs onto it, preventing it from going back up the pore. Of course, the Hsp70 molecule also is preventing improper folding, as in the diagram in (3).
To fulfill their function, the Hsp70 molecules must be able to bind to the hydrophobic regions but also be able to release themselves when they are no longer need. The unbinding is caused by ATP binding and hydrolysis in the ATPase site of the Hsp70.
The presence of heat shock proteins was first discovered in the 1950’s, when a lab technicain accidentally allowed some fruit fly (Drosophila) cultures to become too warm. They were studying the “gene puffing” that happens when genes of the chromosomes in the salivary glands of (Drosophila) are transcribed. Various flies (but also some other invertebrates such as newts) have unusual chromosomes in some of their cells. The cells of the salivary glands of (Drosophila) are very large, so they many copies of the genes. This requirement for more gene copies per cell could have been satisfied by making the cells multinucleate. This is what happens in our skeletal muscle cells, which are large cells; they are multinucleate. But (Drosophila) does it differently. It does what is known as endomitosis. After a round of DNA replication the sister chromatids that are formed are not pulled apart at a metaphase stage. Thus a hundred or more rounds of DNA replication, and non-separation of the sister chromatids, results in chromosomes so thick that they can be seen in the light microscope as individual chromosomes (after some squashing of the preparation!) even in interphase cells. When transcription of a gene occurs the DNA loops out of the (i.e. becomes euchromatin) and along with all the RNA polymerase II and nascent mRNAs looks like a “puff”. Specifically, they are called chromosomal puffs and one can tell what genes are being transcribed by noting where along a given chromosome the “puff” is located. When the fruit flies had been under the
too warm conditions, it was noted that the transcriptional activity of only a few specific genes was increased. No one had ever seen these specific genes (then of unknown function) so active before. They were designated as “heat shock genes” and the proteins they were assumed to encoding for were called heat shock proteins. Only much later were these proteins isolated and their function studied. The heat shock genes were the Hsp70 genes!