Endoplasmic reticulum

The endoplasmic reticulum is the first “organelle” we look at. Organelles are generally membrane-enclosed compartments inside the cell. The nucleus, mitochondria, Golgi apparatus and lysosomes are all examples of organelles. The space between the organelles is called the cytosol. The cytosol plus the organelles is referred to as the cytoplasm. The endoplasmic reticulum is a network of membrane sacks and tubes that are continuous with the outer nuclear membrane and extend throughout the cytoplasm. It carries out a number of functions that are important for cells:

A  Functions of the ER

1) Synthesis and processing of new transmembrane or secretory proteins.

This is one of the main functions of the ER. These proteins are synthesized on the rough endoplasmic reticulum. The rough ER is called “rough” because it is studded with ribosomes.

2) Calcium storage. The SERCA (Smoother ER calcium) ATPases in the ER membrane pump calcium out of the cytosol into the ER. This keeps calcium levels low in the cytosol while the ER becomes a major calcium storage organelle. Calcium is then released by inositol trisphosphate (IP3).

Protein misfolding in the ER is thought to cause calcium leakage. This leakage of calcium eventually damages mitochondria causing apoptosis. If this theory is correct it may explain some chronic wasting diseases.

3) Detoxification of foreign organic compounds

The liver ER contains a protein called cytochrome P450 which activates oxygen so that it can attack and modify (typically inert) hydrophobic organic compounds. The addition of oxygen makes compounds more water-soluble so that they can be eliminated.

4) Synthesis of new membrane

B. Overview of protein processing

Some proteins are meant to reside in the cytosol. They are synthesized on free ribosomes in the cytoplasm. Other proteins are meant to be either transmembrane proteins or proteins that reside inside vesicles. These proteins are synthesized by ribosomes that are bound to the endoplasmic reticulum (rough ER).  Proteins synthesized at the ER go through numerous processing steps before they are mature. Some of these processing steps are carried out in the ER. Further processing is done in the Golgi apparatus. After completion, the final step is to send the proteins to their correct destination.

C. George Palade’s pioneering work showing the progressive processing of proteins beginning in the ER.

Georg Palade was a pioneering cell biologist who was interested in the movement of secreted proteins. Some proteins are made in the cytoplasm but others are made at the ER. We now know that proteins made at the ER (rough ER) are either transmembrane proteins or proteins that reside in vesicles. When he started, it really was not understood how proteins got into secretory vesicles. Palade’s work showed that newly made secretory proteins first showed up in the ER and then went to the Golgi and finally into secretory vesicles.  To demonstrate this he used electron microscopy and a technique called pulse-chase labeling and autoradiography to track newly made secretory proteins.

1) Pulse chase labeling

The desire here is to get a high amount of radioactively labeled protein in a narrow window of time. Cells are exposed to a highly radioactive amino acid for a short period (the pulse). The radioactive amino acid is then washed away and cells are given non radioactive amino acid (the chase). The chase is meant to make the cells quickly end using radioactive amino acids to make new proteins. Cells are then processed at various times after the initial labeling in order to follow where proteins go. The goal is to intensely radiolabel proteins during a short time window and then follow where these labeled proteins were in the cell.

2) Autoradiography

Radioactive emissions can expose film just as light can. In this case, the tissue slices prepared from radioactively labeled cells are coated with a film of photographic emulsion (in regular film or print paper, a plastic or paper is coated with the emulsion). Radioactively labeled proteins expose the emulsion. When developed there will be silver grains or tracks over the region where the radioactive protein was in the cell.

           Synthesis of Transmembrane and secretory proteins

A) The signal sequence

Proteins that are to be synthesized on the ER have what is known as a “signal sequence” at the beginning of the protein. In fact proteins can have a number of sequences that are referred to as signal sequences such as the nuclear localization sequence. Perhaps one could think of the ER targeting signal sequence as Signal Sequence having a capital S. This signal sequence is at the beginning of a protein and is the first part of the protein that is synthesized and exposed as the new protein comes out of the endoplasmic reticulum.

The ER targeting signal sequence consists of a hydrophobic region that can span the lipid bilayer and on either side of the hydrophobic region there can be positively charged amino acids. Whether they come before or after the hydrophobic region is actually quite important as it designates whether the signal is a start transfer or stop transfer sequence (as we will see in a bit).

Following the signal sequence is an AXA cut site that is cleaved by the signal peptidase when the AXA sequence is present. If the signal is not cut then it is called a signal anchor sequence. While the signal is normally at the N terminus it does not have to be. There are signaling sequences well away from the N-terminus that are called internal signal sequences.

B Expression of the signal sequence 

When translation begins, the mRNA sequence is read and the amino acids are linked together. As the ribosome moves down the mRNA and the peptide is assembled it emerges from the ribosome. If the emerging peptide contains the N-terminal signal sequence it will be exposed as the peptide emerges from the ribosome. If there is an exposed N-terminal signal, a sequence of events will be initiated. This sequence will be

C  Binding of the signal recognition particle (SRP) 

When the signal sequence of a newly synthesized protein emerges from the ribosome, the signal recognition particle inserts into the ribosome and blocks any further translation. Stopping translation is important because it prevents the nascent peptide from folding. If it were to start folding, it could prevent the peptide from threading into the translocon (see ahead).

1). After the SRP binds it then takes the Ribosome- SRP complex to the ER membrane where it docks with the SRP receptor on the ER membrane. This will position the ribosome over the channel through the ER membrane called the translocon.

2) After the SRP – SRP receptor hands off the ribosome to the translocon, they are released.

3. Translation then resumes. As the peptide is translated it moves through the translocon – and is further processed based on the presence of start and stop transfer sequences.

3) The Translocon

The translocon is a large structure that contains a central channel or passage through the ER membrane. The permanent walls of the channel are primarily made from three proteins called sec 61 a, b, and g. Another protein called Tram provides a passage way from inside the channel to the lipid bilayer. In addition to structural proteins, there are some enzymes that are also associated with the translocon. These include the signal peptidase that cuts off the N terminal signal sequence and the oligosaccharide transfer complex that transfers the sugar chain from dolichol to the asparagine nitrogen to produce the N- linked sugars on glycoproteins.

Getting from the central pore to the bilayer

The main function of the translocon is to make transmembrane proteins. The proteins start out going through the aqueous pore of the translocon channel. When a hydrophobic segment comes through, it needs to move out from the central channel into the surrounding lipid bilayer. The function of Tram is to provide a doorway that allows the hydrophobic segment of a new peptide to move from the central channel, through the wall of the translocon and out to the lipid bilayer.

4) Protein topology

Protein topology is governed by the arrangement of start and stop transfer sequences. It is important to get the protein oriented correctly in the pore of the translocation. This is determined by the first signal sequence. The first signal sequence is essentially a string of hydrophobic amino acids  becomes a transmembrane segment.  However, there can be positive charged on the N-terminal or C-terminal side of the hydrophobic segment that determine how the signal sequence will be oriented in the pore of the translocon. Positive charges on the N terminal side (i.e. at the beginning of the hydrophobic string) determine this to be a start transfer sequence. If positive charges come after the hydrophobic segement, then it is a stop transfer segment. The string of hydrophobic amino acids will end up in lipid bilayer as a transmembrane segment. The positive charges determines how the first transmembrane segment will bind to the wall of the translocon.

Studies with artificial proteins containing multiple hydrophobic segments have show that even without the charges proteins will be inserted into the membrane correctly. It seems that it is important for the first signal to get oriented correctly and after that, hydrophobic segments will simply alternate in their orientation. So, if the first signal is a start transfer sequence, the second will automatically be a stop transfer sequence and the next hydrophobic segment will start transfer through the translocon again and so forth. 

Signal peptidase

The N- terminal signal will be cleaved if there is an AXA cut site after the hydrophobic segment. If a protein only has the N-terminal signal and it has an AXA cut site, then it will go all the way through the translocon and end up as a soluble protein inside the ER.

Following the rules!

By following the rules – start transfer or stop transfer – and whether to cleave the N-terminal signal or not – one can arrange the topology of a protein in the membrane in a variety of topologies. Compare the following two scenarios. One has the C terminus (COOH) on the cytoplasmic side. The other has the N-terminus on the cytoplasmic side. How were these two different arrangements accomplished? Can you explain how each case follows the rules?

In the following example, we have a protein with both N-terminus and C-terminus on the cytoplasmic side of the ER membrane. Once again, can you explain its arrangement in terms of the rules for stop and start transfer and cleavage of the N-terminus?

Hydropathy plots versus start and stop transfer.

Hydropathy plots show us where there are segments of hydrophobic amino acids along the length of a protein. These segments of hydrophobic amino acids act as start and stop transfer sequences. After the first signal sequence, it is not essential to have positively charged amino acids determining whether the segment is a start or stop transfer sequence. The segments automatically switch from start to stop to start. Note a certain inconsistency in the book where the first (N-terminal segment) is really a stop transfer sequence.

Summary

             Processing of newly made proteins.

Achieving the proper membrane topology of transmembrane proteins is really just the beginning. There are numerous processing steps that can follow.

Processing steps

1. Cleavage of the N-terminal signal sequence (or not). It depends on the presence of an AXA cut site.

2. Glycosylation of proteins.

Proteins can be glycosylated by the addition of sugars. There are two types of glycosylations

A. O-linked

Here, sugars are added to the OH – usually of a serine or threonine. In collagen, an especially modified lysine is formed to give hydroxylysine. Finally, there is one case where sugars are added to the OH of tyrosine. In all cases, sugars are added to an OH group. This is why they are called O-linked. Sugars can also be added to the OH of hydroxylysine. O-linked oligosaccharides are short chains of sugars (from 1 to 4)

B. N-linked

Sugar chains are transferred to the amide nitrogen of asparagine (see below). There is are specific sequences that designate that the sugar chain be transferred to the protein. These are Asn-X-Ser or Asn-X-Thr (in rare instances Asn-X-Cys).

N-linked glycosylation (continued)

Sugar chains are transferred to an asparagine amino acid.

The sugar chains transferred to asparagine are built on dolichol lipids

Synthesis of N-linked sugar chains occurs in two phases.

Phase 1 The sugar chain (i.e. an oligosaccharide) is built on the cytoplasmic side of the ER membrane. Phase 1 is completed when there are the two initial N-acetylglucosamine residues followed by 5 mannose residues.

Phase 2. The dolichol lipid + oligosaccharide is flipped over to the other side of the ER membrane so that the sugars are now on the luminal side of the ER. Here addition of sugars continues until there are nine mannose residues and 3 glucose units are added. At this point, the entire sugar chain is transferred from dolichol to the protein. Subsequently the three glucose units are removed.

Formation of disulfide bonds – the role of PDI

Disulfide bond formation takes place in the lumen of the ER. If disulfide bonds are formed between the wrong cysteines, the protein will be trapped in an incorrectly folded state. An enzyme called protein disulfide isomerase (PDI) breaks and remakes disulfide bonds. If the disulfide bond was not between the right cysteines, this will allow the protein to continued to fold correctly. Once the protein has reached its final folded state, it should place the right cysteines in position to make the correct disulfide bonds and this will help stabilize the protein.

Formation of GPI-linked proteins. 

GPI linked proteins have a signal for cleavage of the protein such that it is transferred over to the glycosylphosphatidylinositol lipid.

Next Stop - the Golgi

             ER to Golgi

B. Proteins collect in vesicles and exit at a few specific sites on the smooth ER Proteins exit in vesicles that are formed by assembly of a coat protein called COP II (Coat protein II or Coatamer II). These exit sites contain large numbers of COPII coated buds. COPII initially drives coat formation but then comes off after complete vesicle formation. After they come off the ER and lose their coats, the vesicles then fuse together to form VTCs (vesiculotubular clusters). Motor proteins (dynein and kinesin) are associated with these VTCs. Dynein is needed to move the VTC along microtubules to the Golgi. At the Golgi, vesicles or VTC get trapped by tether proteins where tethers mediate “long distance” anchoring to the site where vesicles will ultimately fuse. Vesicles will ultimately be targeted to the site of fusion through SNARE-SNARE interactions as we saw for nerve terminal secretion.

ASSEMBLY OF THE COAT

COPII coat assembly depends on a small G protein known as Sar-1. As with other small G proteins, a GEF mediates conversion of the Sar-1 from the GDP bound form to the GTP bound form. The conversion of Sar1 to the GTP-bound form causes it to bind to the membrane  triggers coat assembly.  The coat interacts with transmembrane proteins that accumulate in the buds that form or with receptors that bind to soluble proteins that will be carried in the vesicle.

Disassembly of the Coat

Formation of the coat drives the formation of a membrane but that will ultimate form an independent vesicle when coat assembly is completed. One the coated vesicle forms it soon loses its coat. As mentioned above, Sar-1 in the GTP-bound state drives coat assembly. A GAP protein triggers Sar-1 to hydrolyze its GTP which results in coat disassembly. Ultimately, the energy of GTP has been used to drive formation of a vesicle leaving the ER.

TWO WAY TRAFFIC

So far, we have talked about vesicle traffic going from the ER towards the Golgi

In fact vesicle traffic is bidirectional going from ER=>Golgi and from the Golgi=>ER. The pathway from the Golgi back to the ER is mediated by some different proteins. For one thing, the coat protein is different. COP1 is the coat that generates vesicles going from the Golgi to the ER. In addition, a different G protein called ARF is involved in assembly of the COP1 coats.

Traffic along microtubules

Microtubules in the cells are 25 nm diameter tubes that serve as roadways in the cell. They originate at the microtubule organizing center or centrosome. Microtubules have polarity, their ends being designated as plus or minus. The minus ends are at the centrosome while the plus ends are in the periphery. There are two kinds of motor proteins that move cargo along microtubules, the minus end directed motor known as dynein and the plus end directed motor known as kinesin. The Golgi is usually located near the centrosome where the minus ends of microtubules are located. Movement of vesicular traffic towards the Golgi thus depends on the dynein motor protein. Movement of vesicles back from the Golgi to the ER depends on kinesin.

Microtubule motor proteins (kinesin or dynein) carry vesicles either towards the centr

(c) Tethering

By the time the ER vesicles get to the Golgi, they are uncoated. Uncoating of vesicles takes place when Sar1 hydrolyzes its bound GTP. The uncoating exposes Rab proteins on the vesicle that bind to tether proteins (Golgin) bound to the Golgi membranes.

4) KDEL and the Retrieval pathway

Some proteins are meant to reside in the ER. These proteins contain a signal sequence which marks them as ER resident proteins. There are two signal sequences that designate proteins as ER proteins

1. KKXX at the C terminus of type 1 integral membrane proteins
2. KDEL for ER luminal proteins

Of these, the KDEL signal is better understood. Having a KDEL sequence does not mean that proteins never leave the Golgi. Rather, when they mistakenly go to the Golgi, they are retrieved and sent back to the ER. It is thought that retrieval depends a receptor that binds KDEL and on the pH difference between the Golgi and the ER. The Golgi is more acidic than the ER.

How a difference in pH leads to retrieval of proteins with KDEL

There is a receptor for the KDEL sequence. This receptor binds with high affinity when the pH is low (in the Golgi). This receptor is part of the return pathway to the ER. When the receptor binds to KDEL, it causes both receptor and the KDEL-containing protein to go back to the ER. In the ER, where the pH is higher, the KDEL receptor has low affinity for KDEL and lets go of it. In effect, it drops off the KDEL containing protein in the ER.

Protein Folding and Misfolding in the ER

Protein folding is the most error prone step in the production of proteins. In general, proteins bind to chaperonins until they are properly folded. These ER chaperonins have retention signals that keep them in the ER until they are properly folded.

            -- BIP (binding protein) is one important chaperone protein in the ER

            -- Calnexin and Calreticulin are other examples.      

ER Stress

Alterations in ER homeostasis can cause the accumulation of misfolded proteins. A wide range of cellular environments and events induce ER stress. These include: increased levels of protein synthesis; impaired ubiquitination and proteasomal degradation; deficient autophagy; energy deprivation; an excess or limitation of nutrients; dysregulated calcium levels or redox homeostasis; inflammatory challenges; and hypoxia. When unfolded proteins accumulate in the ER, it triggers the unfolded protein response.

There are three ER response systems that activate when unfolded proteins accumulate in the ER.

1. PERK  PRKR-like ER kinase (PERK)–eukaryotic translation initiation factor 2α (eIF2α)
2. IRE1a Inositol-requiring protein 1α (IRE1α)–X-box-binding protein 1 (XBP1)

3. ATF6a Activating transcription factor (ATF)6α

These three protein systems generally work to accomplish 3 aspects of the unfolded protein response:

            A. Temporary block of proteins synthesis

            B. An increase in protein folding activity

            C. An increase in protein degradation and autophagy

Chronic triggering of the unfolded protein response and inadequacy of the response leads to disease

Chronic ER stress causes a number of problems. One idea is that ER damage or stress leads to loss of calcium from the ER. Mitochondria then take up calcium in an effort to maintain low levels of calcium. However, continued uptake of calcium can lead to release of reactive oxygen species and ultimately, damage to the mitochondria leads to apoptosis. 

3 Examples of disease

A. Neurodegenerative diseases.

B. Metabolic disease

C. Immune dysfunction