4.1: The Endoplasmic Reticulum

Learning Goals

• Explain the structural and functional relationships between the different compartments of the endomembrane system and identify them on different micrographs.
• Describe how proteins are targeted and imported into the endoplasmic reticulum and compare these mechanisms to protein targeting and import into the nucleus.
• Predict the targeting sequences required to insert a protein into the endoplasmic reticulum (ER) membrane in any orientation and predict protein topology from a corresponding domain map.
• Discuss the role of chaperones in protein folding and the role of the proteasome in the Unfolded Protein Response.

Introduction to the Endomembrane System

The endomembrane system consists of the endoplasmic reticulum (ER), Golgi apparatus, lysosomes, and endosomes. These compartments are involved in a great many cellular functions, including the processing of proteins that are destined for export from the cell, dealing with proteins that have been brought in from the outside of the cell, lipid synthesis, and a variety of signaling events.

The endomembrane system has a number of compartments, and cargo travels from one compartment to the next using smaller, membrane-bound structures known as vesicles. We say that the “start” of the endomembrane system is the ER, as this is the point of entry for newly synthesized proteins (Figure 04-01). Once proteins enter the ER they never return to the cytosol; they are carried by vesicle transport to the other compartments of the system. This flow of vesicles is highly regulated. The “end” of the endomembrane is usually considered to be the cell exterior, as proteins that pass through the entire length of this system, without being diverted, will eventually undergo secretion, ending up in the extracellular space, or embedded in the plasma membrane.

Figure 04-01: The compartments of the endomembrane system, and the flow of traffic between them. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

There are three major pathways to travel through the endomembrane system. See if you can trace each one in Figure 04-01.

1. The secretory pathway, which is also often called the “default pathway” through the organelles. It is the path that most newly synthesized ER-targeted proteins will take as they eventually exit the cell. Proteins start in the ER, move to the Golgi and then finally moved to the plasma membrane.
2. The lysosomal pathway, which is the path that newly synthesized digestive enzymes will take as they move to their eventual destination (the lysosome). Proteins start in ER, transported to the Golgi, delivered to the endosomes when they finally go to the lysosome.
3. The endocytic pathway, is the path inward from the cellular exterior for proteins and substances that are unable to cross the membrane through diffusion or by transport protein. Material brought in via endocytosis will travel to endosomes and likely end up in the lysosome, where they can be degraded, and their components recycled into building block molecules. Molecules entering the cell this way are said to have undergone endocytosis.

Regarding: The Nuclear Envelope

It is sometimes said that the nuclear envelope is also a part of the endomembrane system. This is because the outer membrane of the nuclear envelope is continuous with the ER. This means that the space inside the ER, and the space between the membranes of the nuclear envelope (called the perinuclear space) is continuous with the ER lumen. In this textbook we don’t really discuss the nuclear envelope as part of the endomembrane system, even though it is continuous and may get involved in some of the same functions that the ER does. This is mostly to simplify language, as we discuss the various structures and their function.

Proteins that enter this interconnected set of organelles can only enter at the “start” of the pathway. So newly synthesized proteins that are destined for the secretory or lysosomal pathways must always enter at the ER. Material coming into the cell from the extracellular space via endocytosis begins at the plasma membrane. Additionally, proteins that enter the endomembrane system always travel the same route. They don’t skip compartments, or return to the cytosol (unless they are misfolded and need to be destroyed…but more on that later…).

In this topic, we have two main ideas to discuss:

1. How proteins are modified after translation. This is called protein processing.
   • Since protein processing begins in the cytosol and (sometimes) ends in the endomembrane system, we’re going to talk about this first, and then get into the details of how the endomembrane system works.
2. How newly synthesized proteins enter the ER, thus gaining access to the rest of the endomembrane system.

Protein Processing in the Endomembrane System

As you (hopefully!) remember, protein translation happens in the cytosol. (As always, the introduction is there for you to review should you need it.) Once translated, all proteins are processed. Processing can include any number of events, such as the following:

• Folding: All proteins are folded to generate their 3D structure; thus, all proteins are processed in at least this one way. They will form the three (or four) levels of protein structure we mentioned in Chapter 2. The final folded 3D structure is based on the primary sequence of the protein.
• Removal of the first methionine at the N-terminus. Remember that the codon that serves as the “start” codon is also the codon that codes for methionine. In many proteins this methionine is removed once the protein is translated.
• Methylation
• Phosphorylation
• Acetylation
• Formation of disulfide bridges (most common in secreted proteins)
• Glycosylation (only in proteins destined for the cell exterior or the lysosome)
• Cleavage and more…

We will discuss some of these in more detail, but not all. We will focus first on protein folding and how it is managed by the cell. Then we’ll look briefly at how and where disulfide bridges form. Later in the chapter, we’ll look at how proteins are glycosylated, since that process is primarily the job of the Golgi apparatus.

Protein Folding

As the newly synthesized polypeptide emerges from the ribosome, it will immediately begin to fold based on its primary structure. Protein folding is a spontaneous process in which the polypeptide will take on the lowest energy conformation possible. This you should already know from our discussion of membrane proteins in Chapter 2. This is not to say that the protein will spontaneously “find” the proper conformation. Folding is extremely complex, and we continue to learn about it. What we do know boils down to this:

• Proteins can successfully fold a lot of different ways. They do not follow the same path each time.
• It is a process that can go wrong very easily. Not every translated protein ends up properly folded. Misfolded proteins are usually destroyed by the cell.

Figure 04-02, below, perfectly illustrates this concept. In it, we see many of the different steps a protein can take to get from “unfolded” to “properly folded.” Note that this image excludes all of the ways that a protein could fold and end up at a “dead end,” where it would not be able to find the appropriate final conformation. There are just as many, if not more, ways a protein can misfold. Since misfolded proteins are destroyed by the cell, it is unclear exactly how often it happens, but some estimates suggest that as many as 50% of proteins misfold!

Figure 04-02: Some of the different paths a protein can take to become a fully and correctly folded protein. It’s worth noting that this image does not include any of the possible “dead-end” paths, where the protein is unsuccessful in reaching the proper final fold. “ACBP MSM from Folding@home” by Vincent Voelz is shared under a CC-BY-SA 3.0 license and is based on work published by Voelz et al. (2012).

Chaperone Proteins Help Ensure Proteins Fold Properly

The capacity for a protein to spontaneously fold correctly is important for more reasons than you might expect. Improperly folded proteins can be very detrimental to the cell. Not only would the proteins be unable to perform their function, but they might be insoluble and form large aggregates in the cell. In fact, misfolded proteins are a common source of disease, including Alzheimer’s, type 2 diabetes, cystic fibrosis, Parkinson’s, and prion diseases.

To ensure that the highest number of translated polypeptides manage to successfully fold, chaperone proteins often aid in the process (Figure 04-03 and Video 04-01). The new polypeptide forms a complex with chaperones that facilitate folding. Different chaperones help in different ways. Some may help simply by binding to specific regions of the polypeptide (Figure 04-03B) to prevent them from folding too early. This is how a chaperone known as HSP70 works. Others, such as HSP60 (or chaperonin), act as a chamber, providing a protected space away from the rest of the cytosol, where the protein can fold in isolation (Figure 04-03A).

Figure 04-03: Chaperone proteins help proteins fold. (A) HSP60-style chaperone proteins form a chamber, creating a specialized environment for client proteins to fold. The protein enters the chamber, and a chamber cap is placed. When the protein is done folding, it is released. (B) HSP70-style chaperone proteins bind to client proteins as they are synthesized, allowing certain areas to wait before being folded. This allows them to adopt the proper conformation. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Video 04-01: Chaperones and misfolded proteins. The process of protein folding and the role of molecular chaperones HSP60 and HSP70 are discussed.

Misfolded Proteins Are Sent to the Proteosome

It is vital that proteins either fold correctly or get disposed of properly when they misfold. The most obvious reason for this is that misfolded proteins are unlikely to function correctly, and this is true. However, it is equally important that these dysfunctional proteins get sent to the proteasome for degradation. The reason is that a misfolded protein is more likely to form complexes with other misfolded proteins nearby. This is due to exposed nonpolar regions on misfolded proteins that would otherwise be hidden away inside the protein. Misfolded proteins often form large clumps, or aggregates, inside the cell. The result of this is a giant blockage of protein inside the cell that gums up the works and inhibits function. If the aggregate is big enough or sticks around for a long time, it can actually kill the cell. There are a number of diseases in which protein aggregates accumulate in cells. For example, in Alzheimer’s disease and other forms of dementia, large protein aggregates are observed inside neuronal cells. One hypothesis is that these aggregates disrupt cellular function, and the cells die as a result. In sickle-cell anemia, the hemoglobin proteins misfold due to a mutation and form large clumps that ultimately destroy red blood cells.

The proteosome is a large complex in the cytosol of the cell whose sole function is to destroy proteins that are damaged, misfolded, or no longer needed. It is one of the larger protein complexes inside the cell.

Want to compare the size of the proteosome to other cellular proteins? Follow this link to the Protein Data Bank interactive map!

How does the proteosome work? Proteins that need to be degraded are tagged with a small peptide tag known as ubiquitin (Figure 04-04 and Video 04-02). The ubiquitin tag is recognized by other proteins, whose task is to deliver the ubiquitinated protein to the proteosome. Once there, the protein is threaded into the interior of the cylindrical center of the proteasome, and the peptide bonds are broken in a chemical process known as proteolysis. That way, the amino acids in the protein are kept and can be recycled by the cell. The ubiquitin is also removed and recycled.

Figure 04-04: Degradation of ubiquitin-tagged proteins by the proteosome. Ubiquitin attaches to proteins, which sends them to the proteasome. The protein enters the chamber of the proteasome and is digested. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Video 04-02: The proteasome: the cell’s trash processor in action

Protein Folding and the Formation of Disulfide Bridges

Disulfide bridges are covalent bonds that form between the sulfurs of cysteines in an amino acid chain. Usually, the cysteines are far away from each other in the linear polypeptide chain but are brought together during folding. Enzymes usually facilitate the formation of disulfide bridges (also known as S-S bonds). These bonds help stabilize the 3D structure of a protein and/or help hold different subunits together. We touched on them briefly in Topic 2.3 as well, when discussing protein folding.

Interestingly, disulfide bridges only form under very specific conditions. They can only be formed under oxidizing conditions within the cell. (Hint: Remember your general chemistry on redox?) In reducing environments, they are unstable and tend to fall apart. What is the most interesting about this is that the cytosol tends to be a more reducing environment, which means that disulfide bridges are not easily formed or maintained there. On the other hand, the interior of the ER, known as the ER lumen, and the extracellular space tend to be oxidizing environments. This means that proteins that have moved through the ER during folding are far more likely to have disulfide bridges in them. We’ll see more on which proteins are folded in the ER and why a little bit later in this topic, but in a nutshell, proteins that are destined for any compartments of the endomembrane system or proteins that are targeted to the plasma membrane and/or the exterior of the cell are the ones that enter and do at least some of their folding in the ER.

The ER Is the Point of Entry into the Endomembrane System for Newly Synthesized Proteins

The ER consists of flattened membrane sacs, known as cisternae, and tubules. It is directly connected to the outer membrane of the nuclear envelope, but unlike most of the cartoons of the ER found in textbooks like this, it stretches throughout the entire cell. There is no part of the cell that is far from the ER. The ER has a number of different regions in it that are all connected together (Figure 04-05):

• Rough ER (rER). The cytosolic surface of the rER membranes has docked ribosomes that are synthesizing proteins for import into the ER. This is the site of synthesis for proteins destined for secretion, lysosomes, or membranes.
• Smooth ER (sER). This is continuous with rough ER, as shown in the photo below, and is the site of lipid and steroid synthesis. As mentioned in Chapter 2, new lipids and membranes are made in the sER.
• At the site where these two types of ER meet, there is a third form of ER known as transitional ER (tER). This is the site where vesicles usually form and newly synthesized proteins exit the ER in order to move on to the next destination (i.e., the Golgi apparatus). As such, this region is also known as an ER exit site.

Figure 04-05: Electron micrograph showing the ER and Golgi in a pancreatic cell of a guinea pig. The rough ER (rER) can be identified by its “rough” appearance due to the ribosomes docked on the surface of the membrane. The smooth ER (sER) has no ribosomes but still looks like it should be a bunch of interconnected tubules that have been cut in cross section. The point at which they join and the ER transitions from rough to smooth is known as transitional ER (tER) and is an exit point for vesicles leaving the ER. The Golgi apparatus of the cell is also visible here and can be identified by the fact that it resembles larger sacs that are somewhat flattened. Original micrograph: George E. Palade (2012), CIL:37239, Cavia porcellus, pancreatic cell. CIL. Dataset. doi.org/10.7295/W9CIL37239. Labeled by Robin Young. Licensed by CC BY-NC-SA 3.0.

There are a couple of things to note about the traffic between the ER and the cytosol:

• The ER is the point of entry into the entire endomembrane system for newly synthesized proteins, so there is a lot of traffic heading from the cytosol to the ER.
• On the other hand, once a protein enters the ER, it does not return to the cytosol.
• Proteins that need to leave the ER to travel to the next compartment in the endomembrane system (which is the Golgi) will get packaged into vesicles, which will bud from the tER, and then fuse with the Golgi membrane. This way the protein can travel between compartments without returning to the cytosol.

Misfolded Proteins Leave the ER and Are Degraded in the Cytosol

There is one notable exception to the flow of protein traffic from the ER to the rest of the endomembrane system. Misfolded proteins that cannot be saved by the chaperone proteins must be sent to the proteasome for degradation. Since the proteasome is in the cytosol, this is the one instance when a protein will be transferred back across the ER membrane to the cytosol.

Just like in the cytosol, an accumulation of misfolded proteins in the ER lumen triggers the production of chaperone proteins and the expansion of ER, which can help reduce misfolding and aggregation. This is known as the unfolded protein response (UPR). Dealing with improperly folded proteins is thought to be a massive undertaking in the cell, which gets much worse when mutations exist that result in proteins that don’t fold efficiently.

Many proteins with multiple subunits, such as antibodies, are assembled in the ER. If these proteins are not properly assembled (e.g., via the formation of disulfide bridges), they will also trigger the UPR to address it.

Cells make lots of mistakes in the assembly of proteins. Proteins are made of hundreds, if not thousands, of amino acids, making them easy to misfold or misassemble. A huge part of the job of the ER is to ensure that properly folded proteins move on and misfolded ones are dealt with efficiently and don’t build up inside the cell and block traffic.

Entry/Exit from the ER Is Strictly Controlled

Just like the nucleus, the cell controls precisely what is allowed to enter and exit the ER. However, since the role of the ER is different from that of the nucleus and its structure far more intricate, controlling access is also more complex. Like the nucleus, only proteins with the proper targeting sequence will be allowed to enter the ER. Of those that are allowed to enter, some will become residents of the ER, while others will simply be passing through on their way to other destinations farther along in the secretory or lysosomal pathways.

Proteins Are Inserted into the ER Co-translationally

This is our second organelle in which we discuss protein import. Our first example was nuclear import (in Chapter 3). In the next chapter (Chapter 5), we will see how proteins enter the mitochondria and chloroplasts. In the case of the endomembrane system, there is more than one process to consider, as several organelles are included. Each compartment will have its own unique sequences, which must be properly read at the correct moment in the pathway. The additional sequences will be discussed later, when we explore the function of each of the compartments.

The first thing you must know is that translation of all proteins, including ER proteins, begins on free ribosomes in the cytosol (Figure 04-06). Whether or not a ribosome settles on the rER or stays in the cytosol to complete translation is dependent entirely on the protein it is currently translating. A ribosome that is currently translating a protein destined for the ER will attach itself to the surface of the rER during translation through a series of cues found in the amino acid sequence of the new protein. It is this field of ribosomes actively translating on the surface that gives the ER it its “rough” appearance. Smooth ER (sER) does not participate in protein synthesis (sER is important in lipid synthesis as well as metabolizing many toxic chemicals, like ethanol).

Figure 04-06: All proteins are translated by the same pool of ribosomes in the cytosol. As the ribosomes bind to the mRNA (blue thread), they begin translation (emerging green line). If they contain an ER insertion sequence, they are taken to the ER membrane. However, if no such sequence is present, the ribosomes continue to translate the protein in the cytosol. Once translation is finished, the ribosomes separate and are free in the cytosol. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Structurally, the proteins entering the ER fall into two major categories:

1. Soluble proteins: These proteins are completely translocated across the ER membrane into the ER lumen. They do not contain any membrane-bound portions.
2. Integral membrane proteins: These proteins are only partially translocated into the ER and end up getting “stuck” with part of the protein embedded inside the membrane. These proteins may be destined for the ER, membranes of another organelle (Golgi, lysosomes, or endosomes), or the plasma membrane. Once a protein is inserted into a membrane, it cannot be removed.

The difference between the formation of a soluble protein and a membrane-bound protein is due to the number and placement of the ER insertion sequences. These sequences are used to identify when the ribosome should dock on the ER membrane and also which regions should become transmembrane domains. Here are some things to know about ER insertion sequences:

• Unlike the nuclear localization sequence (NLS), the specific order of amino acids in an insertion sequence is not as important as the chemical properties of the amino acids within the sequence. In all cases, the ER insertion sequence is about 8–10 nonpolar amino acids in a row.
• These ER insertion sequences go by a variety of names, depending once again on their location in the protein.
  • If the sequence is directly at the N-terminus, it is called the signal sequence, the signal peptide, or an N-terminal START sequence.
  • If the sequence is anywhere else within the primary sequence, it will be called an internal START or STOP transfer sequence, depending on how it aligns with the other ER sequences in the polypeptide.
• Despite the different names and different locations, the sequence itself remains the same. It is still 8–10 nonpolar amino acids, as mentioned above. The location of these targeting sequences within the polypeptide chain determines whether a protein is soluble or an integral membrane protein and also impacts the structure and orientations within the membrane.

Figure 04-07: Insertion of a protein into the ER. Insertion requires an ER targeting sequence, the signal recognition particle (SRP) receptor, and translocation channel. The protein is recognized by the sequence and brought to the translocation channel to be inserted into the ER lumen. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Insertion into the ER takes place in a number of steps and is illustrated in Figure 04-07:

1. An ER insertion sequence is translated and almost immediately recognized by a ribonucleoprotein known as the signal recognition particle (or SRP).
2. The SRP binds to this sequence and inhibits translation.
3. The entire complex (ribosome + mRNA + partially translated protein) is brought to the ER and binds to a special SRP receptor protein in the ER membrane. The ribosome becomes attached to a translocation channel for the newly synthesized polypeptide. This attachment is facilitated by the SRP receptor and requires GTP as an energy source.
4. As the ribosome becomes attached, the SRP is removed and translation resumes, but now the new protein is being pushed through the translocation channel into the ER lumen.

Video 04-03 does an excellent job of showing the ribosome docks onto the ER at the molecular level. However, it has no narration, so you’re going to have to consider when each step is happening on your own.

Video 04-03: Initiation of protein insertion into the ER via the translocation channel.

Different Types of Protein Insertion into the ER Membrane

Since both soluble and membrane-bound proteins are inserted in this way, using these same insertion sequences, it stands to reason that we need to explore a few different scenarios. In all cases, the location and order of the various ER insertion signals will determine whether a protein is soluble or membrane bound as well as how many times it passes through the membrane. Let’s explore how this works.

Soluble Proteins

Soluble proteins need only a single transfer sequence, and it is always found at the N-terminus (Figure 04-08). After the signal sequence is recognized, the ribosome docks, and the polypeptide is threaded through the translocation channel into the lumen of the ER as it is synthesized. The signal sequence remains embedded in the membrane and is later cleaved off by a protein called the signal peptidase. Once that happens, the new protein is free and soluble in the ER lumen.

Figure 04-08: Insertion of a soluble protein through the ER membrane. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Membrane Proteins

A key point in the production of membrane proteins is that the orientation of a protein in the membrane is established when it is first inserted into the membrane, during translation. The orientation of the protein persists throughout its life-span, even as it is shuttled from one compartment to the next. That is, the cytosolic side of the protein remains on the cytosolic side of the membrane throughout its entire life. More on this later.

As membrane proteins are being translated, ribosome docking and co-translational insertion will not begin until an ER insertion sequence is encountered. Thus, the very first insertion sequence encountered is called the “START transfer” sequence. The first START may be at the N-terminus, as we saw in the previous example, but it doesn’t have to be.

The new protein will continue to be translated into the ER until a stop codon is reached (which ends translation) or a second insertion sequence is encountered. The second insertion sequence serves as a “STOP transfer” signal, which will close the translocation channel, release the ribosome, and stop co-translational insertion. When the START and STOP sequences are inside the amino acid sequence (i.e., not at the N-terminus), they serve as transmembrane domains for the growing protein. If the ribosome is still translating the protein after the STOP is encountered, it will remain tethered to the ER via the translating protein until either a new START sequence is met or translation ends. This process can be repeated many times in a single protein for as many ER insertion sequences as exist in the primary structure.

To further clarify how the ER insertion sequences are used to create membrane proteins, we have provided a number of examples.

Example #1

A single internal START sequence produces a protein with one transmembrane domain and the N-terminus on the cytosolic side.

Figure 04-09: Insertion of a protein with one internal START sequence. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

In this example (see Figure 04-09), the internal ER insertion sequence is recognized partway through protein synthesis, and the ribosome is brought to the translocation channel. That sequence is the START transfer, and everything after it is threaded through the translocation channel. Once translation is finished, the ribosome is released. The translocation channel opens and allows the protein to diffuse laterally into the membrane. The internal START transfer sequence is not cleaved and instead remains embedded in the membrane, becoming the transmembrane domain for the protein. As discussed in Chapter 2, the transmembrane domain of the protein holds the protein in the membrane because of the very strong association between the nonpolar amino acids in this region and the nonpolar lipid tails in the lipid bilayer. The N-terminus remains in the cytosol, while the C-terminus ends up inside the ER lumen.

Example #2

An N-terminal START transfer sequence followed by a STOP transfer creates a single-pass protein with an N-terminus in the lumen and a C-terminus in the cytosol.

Figure 04-10: Insertion of a protein with an N-terminal START and a STOP insertion sequence. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Similar to the soluble protein example (Figure 04-08), this next example protein has an N-terminal START sequence (Figure 04-10). This targets it immediately to the ER membrane, and the growing polypeptide is threaded through the channel from the very beginning of translation. At some point during translation, it runs into another ER insertion sequence, which will act as a STOP transfer sequence. When the STOP transfer sequence is encountered, it causes the translocation channel to stop threading polypeptide through the translocation channel into the ER lumen. The ribosome undocks from the membrane but is still tethered via the translating protein. Since there are no other insertion sequences in this protein, the ribosome will complete translation undocked but tethered. Since the START sequence is N-terminal, it will be cleaved off, which results in the N-terminus of the protein being free in the ER lumen, while the C-terminus remains in the cytosol.

Example #3

An internal START followed by a STOP creates a double pass membrane protein with both an N-terminus and a C-terminus in the cytosol.

Figure 04-11: A protein with two transmembrane domains that has been inserted into the membrane. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

In this example (Figure 04-11), the first insertion sequence encountered is in the middle of the polypeptide, so it will be an internal START. Again, the internal START sequence initiates ribosome docking, and the polypeptide begins to thread through the translocation channel. This continues until the second insertion sequence is encountered (a STOP transfer sequence). The STOP sequence ends insertion through the translocation channel, and the ribosome once again completes translation undocked but tethered to the ER. Once translation is complete, the START and STOP are released from the translocation channel and diffuse laterally into the membrane. In this case, both insertion sequences are retained and become transmembrane domains. Both the N- and C-termini of the resulting protein will be in the cytosol.

Example #4

A protein with many membrane-spanning regions.

Using the three previous scenarios as a foundation, you can create a protein with any number of transmembrane domains simply by adding more insertion sequences, which become alternating START and STOP transfer sequences. A START transfer sequence will bring the ribosome to the translocation channel to thread the growing polypeptide into the lumen, and the STOP transfer sequence will release the ribosome, thereby ending the continued use of the translocation channel. The internal START and STOP transfer sequences will each become transmembrane domains, whereas an N-terminal START (if present) will get cleaved. In this way, the protein is essentially stitched into the membrane, and the ribosome is bound and released a number of times. Our final example (Figure 04-12) has six transmembrane regions with the N- and C-termini in the cytosol.

Figure 04-12: Multispanning membrane protein with START and STOP sequences noted. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

We hope that going through these examples has helped make sense of how protein ER insertion sequences work. To briefly summarize, we want you to take away the following the key points from this discussion of protein insertion into the ER:

• The ER insertion sequence is always 8–10 nonpolar amino acids. The difference between how each of the sequences is treated by the cell is due entirely to its location within the primary sequence of the growing polypeptide.
• There are two major categories of hydrophobic signals used in the insertion of membrane proteins. All of these are membrane-crossing domains:
  1. START transfer sequences. There are two kinds of start transfer sequences:
     • N-terminal START transfer sequence. Like its name says, this sequence is at the N-terminus of the protein. It remains in the membrane during translation and is cleaved off of the protein by the signal peptidase. This is also called the signal peptide or signal sequence.
     • Internal START transfer sequence. Similar to a signal sequence but located internally (i.e., not at the N-terminal end of the protein). It also binds to the SRP and initiates transfer into the ER. Unlike the signal sequence, it is not cleaved after transfer of the protein.
  2. STOP transfer sequence. A STOP transfer is never the only signal in a polypeptide chain. It follows either an N-terminal or an internal START transfer sequence. The STOP transfer signal is a membrane-crossing domain. It remains in the membrane. The sequence is not cleaved.
• The orientation of the protein in the membrane is completely dependent on whether there is an N-terminal START sequence or not. If the protein has an N-terminal START, then the N-terminus of the protein will be in the ER lumen. If the first ER insertion sequence encountered is internal, then the N-terminus will remain on the cytosolic side of the ER membrane.
• The number of transmembrane domains a given protein will have will be equivalent to the number of internal transfer sequences (i.e., all STARTS and STOPS that are anywhere other than at the N-terminus).

Video 04-04 is an animation that does an excellent job of showing the details of protein insertion into the ER. We find that students really benefit from seeing this concept in action.

Video 04-04: Several scenarios of protein import into the ER. Basic principles and several example scenarios are shown.

ER Resident Proteins Require an Additional Signal to Stay in the ER

ER resident proteins are proteins that are retained in the ER, as this is where they function. The chaperone proteins that are sometimes required inside the ER to help with protein folding are a great example of an ER resident. These proteins are inserted into the ER as usual, using the ER insertion sequences in their primary sequence, but also carry an ER retention signal called the KDEL (or HDEL in some species) sequence. It is named the KDEL sequence because this is the one-letter code for each of the amino acids in the sequence. It is located in the primary sequence of the protein, always at the C-terminus. The KDEL sequence ensures that any ER resident proteins that might accidentally get packaged into vesicles and shipped to the Golgi will be captured and sent back to the ER.

Protein Insertion and Membrane Asymmetry

As you may recall from Chapter 2, membranes are asymmetric, meaning that the cytosolic side is different from the noncytosolic side. Part of this membrane asymmetry is provided by the location and orientation of the proteins in the membrane. The initial insertion of these proteins into the ER membrane is a big part of how that asymmetry is established. Once that protein is inserted into the membrane, it can’t be removed, so it needs to be done right.

It is also very important that proteins remain properly oriented in the membrane as they move through the organelles of the endomembrane system toward their final destination. The use of membrane-bound vesicles is ideal for this, as the method of budding and fusion of vesicles maintains the orientation of the protein in the membrane while also eliminating any possibility of release into the cytosol (Figure 04-13). In all cases, and at every step, the cytosolic side of the membrane faces the cytosol. This is a key concept in understanding the endomembrane system. Check out the protein orientation in Figure 04-13 and notice that the part of the protein pointing into the cytosol is always the same part even when it gets to the donor compartment.

Figure 04-13: Vesicular traffic maintains the orientation of proteins in the membrane. This image was created by Dr. Robin Young and is shared under a CC BY-SA 4.0 license.

Putting it into Practice: Mapping Protein Domains

As we learned in Chapter 2, bioinformatics is a useful tool to identify known patterns within a protein sequence. We also explored how hydropathy plots allow us to identify potential transmembrane domains based on the amino acid sequence of the protein. If we add that to the information you’ve learned about so far in this chapter, we get a clearer picture of how we can learn about proteins using bioinformatics. You now know that when a hydropathy plot identifies hydrophobic sequences, it’s also identifying the ER insertion sequences that exist within the amino acid sequence. Hopefully, this shows you how the signatures of many functions of proteins exist in their amino acid sequences. Other sequences, such as the NLS or the KDEL, would also be easily recognized by an algorithm that was created to search for them. Import sequences are a great example, but they are by no means the only patterns or motifs that we can identify in a sequence that tell us something about protein function. Regions of a protein that have a specific function attributed to them are called protein domains.

With all of the different patterns and motifs within proteins, scientists need a system to keep track of which motifs exist in any given protein based on the bioinformatic analysis. Visual representations of the linear protein chain are used to map signature motifs within proteins. These are known as domain maps. Figure 04-14, below, shows how we could collect the hydropathy plot data from a given protein (Glycophorin A) and translate them into a domain map that shows how these regions function. Glycophorin A is a membrane protein found in red blood cells (see Figure 02-18, found in Topic 2.3).

Figure 04-14: Domain mapping of Glycophorin A using information from its hydropathy plot to identify ER insertion sequences. (A) Kyle and Doolittle hydropathy plot generated using Expasy Protscale, based on Glycophorin A sequence data from UniProt. The y-axis indicates the hydrophobicity score (see Chapter 2 for tips on interpreting this graph). The x-axis indicates the amino acid number. (B) Domain map for Glycophorin A based on hydropathy plot data in A. (C). Diagram of final protein and how it will be inserted into the ER membrane during co-translational insertion. Since this protein has no other identified targeting sequences, its final destination will be the plasma membrane. This image was created by Dr. Robin Young and is shared under a CC BY-SA 4.0 license.

The hydropathy plot of Glycophorin A (Figure 04-14A) shows two regions within the amino acid sequence that have high hydrophobicity. These two regions are the two ER insertion sequences. Using the information in this hydropathy plot, we can draw a domain map for the protein (Figure 04-14B). The first hydrophobic region is directly at the N-terminus, which makes it an N-terminal START sequence. The second is in the interior of the protein, and it follows a START, so it is a STOP transfer sequence, and it will become a transmembrane domain. From the domain map that we have created, we can also draw the predicted protein and its orientation in the ER membrane (Figure 04-14C). Since the orientation of the protein is preserved as it moves through the endomembrane system (Figure 04-13), you should also be able to predict the orientation of the protein in its final destination, which would be the plasma membrane in the case of Glycophorin A. The portion of the protein that is currently in the ER lumen will eventually be placed on the exterior side of the plasma membrane. The cytosolic side of the protein always remains on the cytosolic side.

For this example, we have focused specifically on mapping ER insertion sequences. However, it is important to remember that there are lots of things that can be mapped in this way. For example, you can map all of the different localization sequences (i.e., NLS, NES, ER insertion, KDEL, etc.) onto domain maps. If the protein was an enzyme with an active site that could be identified, then that could also be mapped. Many secreted proteins also have sites where they get glycosylated. Glycophorin A (the example from Figure 04-14) is glycosylated multiple times. If you were to follow the link to the bioinformatic protein data, the glycosylation sites are also identified and mapped. Maps of this kind are very important for helping us visualize and understand the structure and function of the proteins we study. You will likely have the opportunity to practice domain mapping as we continue to work through the various organelles and how proteins are targeted to them.