Table 04-01: Comparison of the components of the three “canonical coats” covered in this textbook

Comparing Vesicle Coatings
Coat	Coat components	Adaptor	GTPase	Scission protein
Clathrin	Clathrin triskelion complex	Adaptins (AP1, AP2, or AP3) or GGA complexes	Arf (for GGAs only)	Dynamin
COPI	7 subunits in complex	← Included in coat	Arf	Included in coat
COPII	Sec13/31 complex	Sec23/24 complex	Sar1	Included in coat

Clathrin-Coated Vesicles: A Case Study

Since clathrin is the most extensively studied of the vesicle coats, we will use it as a case study to look at the finer details of how vesicle coats form. The clathrin cage is made from two different kinds of proteins known as the heavy and light chains. These chains come together in a specific arrangement to form a triskelion, which can be seen in Figure 04-18.

The triskelions bind to the adaptor (usually adaptin for clathrin-coated vesicles) and assemble to form the coat. As they assemble, they pull the membrane with them to form a ball. While the lattice helps shape the vesicle, it cannot cut the vesicle off from the donor membrane. A separate scission protein called dynamin is used for this purpose. Dynamin has a spiral shape and wraps around the stalk formed by the budding vesicle. Dynamin binds GTP and hydrolyzes it, which allows the protein to constrict around the membrane stalk until the membrane splits and the vesicle is released.

Clathrin works primarily to mediate traffic between the TGN, the endosomes, lysosome, and the plasma membrane. It’s important to note that not all traffic in this area uses clathrin, but most of the traffic that clathrin mediates is somewhere in this region. For example, clathrin is used in certain kinds of endocytosis, which means that the cargo is coming in from the cell exterior. It’s also used to mediate the movement of newly synthesized lysosomal proteins as they move from the TGN to the endosome, which will eventually mature into the lysosome. A notable exception is that clathrin is not usually involved in protein secretion (i.e., the default pathway out of the cell).

COPI and COPII Coats

The process of vesicle budding is essentially the same in COP-coated vesicles as it was in the clathrin-coated vesicles we just saw. In fact, all three follow the same general trajectory that we saw at the start of this section. Video 04-05 (shown below) is an excellent molecular animation of COPII vesicle formation. The different structures of the coats make for differences in the geometry of the cage that is formed and the size of the vesicle (Figure 04-19). Additionally, COPI and COPII coats both are able to promote scission themselves. As such, no additional scission protein (like dynamin) is required. Instead, a small GTPase is used as part of the coat and helps the coat release when its job is done. For COPI, the GTPase used is known as Arf, and for COPII, it’s called Sar1.

COPI and COPII mostly mediate traffic between the ER and Golgi and travel in opposite directions (Figure 04-20). This was mentioned briefly above, but this list gives you more detail:

• COPII vesicles move in the forward direction from the ER to the Golgi (i.e., anterograde traffic). As such, it usually is transporting newly synthesized proteins that need to leave the ER on their journey through the endomembrane system.
• COPI vesicles, on the other hand, mediate retrograde traffic back to the ER from the Golgi. This will include proteins that need to be returned to the ER and that are residents of that compartment. This is where the ER retention signal (also known as KDEL, first discussed in Topic 4.1) becomes important.
  • The KDEL is recognized by the KDEL receptor, a transmembrane protein that lives in the Golgi where vesicles from the ER fuse.
  • The KDEL receptor binds to the KDEL sequence, which is a part of the ER resident protein primary sequence.
  • When the KDEL receptor binds to its cargo, a conformation change happens that exposes a COPI binding site on the cytosolic side of the protein.
  • Once the COPI binding site is exposed, the COPI coat can assemble so that the KDEL receptor, with its cargo, can be packaged into vesicles and sent back to the ER.
  • Once its cargo is released, the empty KDEL receptor is returned to the Golgi via COPII-coated vesicles.

Interestingly, COPI and COPII coats both have a unique additional feature, which we really only discovered in the last 10–20 years. The COPI and COPII cages are somewhat flexible in how they are arranged on the membrane. As such, they can produce larger “vesicles” that can accommodate cargo that would not otherwise fit. For example, collagen is one of the most abundant proteins in the extracellular matrix of animals, which we were introduced to briefly at the end of Chapter 2 (see Figure 02-20). Collagen is synthesized by the cell in the same way that other secreted proteins are: it is co-translationally inserted into the ER lumen, travels through the endomembrane system, and then is packaged into vesicles at the TGN to be sent to the plasma membrane for secretion. Collagen is a very large, fibrous protein, so it doesn’t fit into traditional COPII vesicles. However, COPII is able to rearrange itself into a giant tubule that is large enough to accommodate collagen so that it can be sent out to the extracellular matrix.

Other “Coat” Proteins

As mentioned earlier, our focus in this textbook is on the more traditional vesicle coats that do the bulk of the work in the endomembrane system. However, there are a number of other vesicle “coats” that are involved in the formation of vesicles that are often ignored in introductory cell biology texts such as these. We will list them here, and what they do, so that you have a more complete perspective of vesicle traffic. Also keep in mind that this list might not be complete. Like so many aspects of cell biology, this is an area of active research, and there’s always more to know!

• Caveolin helps create vesicles during endocytosis. Whereas clathrin-coated vesicles are usually used for receptor-mediated endocytosis, caveolin is used for a type of endocytosis known as pinocytosis. We’ll learn more about endocytosis (but not caveolin) in the next topic in this chapter.
• The SNX/retromer complex is primarily involved in retrograde traffic from the endosome to the TGN. In general, it forms large tubules that help in cargo selection. Then the vesicles are budded off the end of the tubule.
• ESCRT is pretty cool, as it is used to bud vesicles in a way that is opposite from most other coats. Instead of budding vesicles into the cytosol, they bud them away from the cytosolic compartment. This is usually used to push vesicles into the endosome so that membrane proteins can be fully degraded by the lysosome. Unfortunately, ESCRT is a common target for hijacking by membrane-bound viruses to help them bud out of cells so that they can go move to a new cell and restart the infection cycle. Both HIV-1 and the Ebola virus are known to do this.

While their mechanism of function is different from the more canonical coats, a few truths still hold. These protein complexes help bend the membrane and pinch off the new vesicle. The retromer and ESCRT also have mechanisms to choose cargo, whereas caveolin is considered nonspecific.

Step 2. Transport of Vesicles from the Donor Compartment to the Target Compartment

There are two possible modes of transport for a vesicle:

1. Over short distances, vesicles can move by diffusion. This is thought to be quite common in mitosis of plant cells, especially when a new cell wall needs to be rapidly secreted between the two new cells. It is also known as bulk flow.
2. Over longer distances, vesicles move along cytoskeletal tracks (usually microtubules but also actin) and are moved by motor proteins (kinesins or dynein for microtubules, myosin for actin).

As mentioned before, we will look at how the cytoskeleton works in Chapter 6, so we direct you there to understand how this process might work.

Steps 3 and 4. Targeting of Vesicles (a.k.a. Docking and Fusion)

Like vesicle budding, docking and fusion must be specific. Errors cannot happen or the cell might die. As such, there is additional machinery involved in making sure that docking and fusion happen accurately and efficiently at each of the different target membranes.

3. Vesicle Docking

Vesicle docking is a way of bringing a vesicle in close to the target membrane to place it in the perfect position for the final fusion step (see Figure 04-21). There are two main categories of proteins involved in this process: Rabs and tethers.

• Rabs are small GTPases that sit on the vesicle surface when activated and help identify the vesicle as one that is headed to a particular target membrane.
  • Rabs are said to be used for “membrane identity.” This means that each organelle in the endomembrane system has its own sets of Rabs. If a vesicle buds from the ER, for example, any ER Rabs will be lost, and a vesicle Rab will dock. Once the vesicle fuses with the target compartment, the vesicle Rab will fall off, and a new Rab will take its place.
  • Rabs are part of a larger “superfamily” of small GTPases whose members you will meet over and over again in cell biology. Ran, which you may remember from nuclear import in Chapter 3, is also part of this family, as well as Arf1 and Sar1, which help with vesicle coat assembly and disassembly in COPI and COPII, respectively. Even tubulin (used to make microtubules) is a distant relative.
• Tethers are a much more diverse group of proteins, usually with very little genetic similarity between them. However, their role is the same in all cases: to bind to the vesicle Rab and help capture the vesicle from the cytosol and pull it in closer to the membrane of the target compartment.

4. Fusion with the Target Compartment

Once the Rabs and tethers have done their work, the final step of this process is mediated by a family of proteins known as SNAREs. The word SNARE stands for SNAP receptor (SNAP itself is an acronym that stands for synaptosomal-associated protein). For this reason, we always write the name of the protein in all capital letters as SNARE.

SNAREs are generally categorized into two major groups: vesicle-SNAREs (v-SNAREs) and target-SNAREs (t-SNAREs). This grouping is based on their location in the cell. The v-SNARE is embedded in the vesicle membrane, and the t-SNARE is embedded in the target membrane.

Structurally, SNAREs fall into two major categories: Q- and R-SNAREs. In order for membrane fusion to occur via SNAREs, one R-SNARE and 3 Q-SNAREs must be present. Some act as v-SNAREs, whereas others will be t-SNAREs.

Once the vesicle has been tethered, the four SNARE coils interact and “zipper” together (Figures 04-21 and 04-22). This pulls the vesicle in tightly enough that all of the water molecules get pushed out of the way, and the two membranes can interact directly. Once that happens, the membrane lipids can intermingle, and the membrane will fuse. Both v-SNAREs and t-SNAREs must be present for this to occur. This binding is very specific…not any old v- or t-SNARE will do.

A further illustration of vesicle fusion can be seen in the Video 04-06. At the end of the video, a protein comes in and uncoils the four SNAREs from each other. This is known as resetting the system, which was mentioned way back at the start of this topic. Separating the SNAREs after fusion is vital so that they are available for the next vesicle that docks.

In addition to uncoiling the SNAREs, resetting the system involves transporting the v-SNAREs back to their original target compartment so that they can be used in another round of vesicle fusion. Transporting SNAREs back is not trivial, as you need to make sure that they don’t accidentally get used in transport. Usually, the SNAREs that are traveling as cargo get covered up by a regulatory protein (called n-Sec1) so that they can’t get in the way. But that story is mostly beyond the scope of this textbook.

Topic 4.3: The Golgi Apparatus
The Heart of the Endomembrane System

Introduction

As material travels through the endomembrane system, it will move from one compartment to the next, using vesicles to traffic between compartments. There is an order to the flow of traffic within this system. Newly synthesized proteins always start their journey at the ER, which was our focus at the start of this chapter. Then they are packaged into vesicles and are sent to the Golgi apparatus. From there, they may take different paths, depending on their role and destination. Some proteins will be sent to the endosome and then the lysosome, while others will head directly for the cell exterior. Even cargo that has entered the cell via the endocytic pathway (Figure 04-01) sometimes comes as far into the cell as the Golgi before being redirected to another destination.

Just like the ER, and the other organelles of the cell, the Golgi apparatus has multiple roles. It is often considered to be the heart of the endomembrane system, as it receives cargo from all directions (ER, plasma membrane, endosomes), modifies it, and then repackages it into new vesicles to be sent to new destinations. It is also the site of all of the major cellular work involving polysaccharides. While some of the work starts or ends elsewhere in the cell, the Golgi is the site of virtually all of the known glycosyltransferases (i.e., enzymes capable of covalently attaching sugars to each other). Not only is this important for the production of glycoproteins (i.e., proteins with sugars attached), but it is vital for the production of the cell walls in plants, algae, and fungi. In this topic, we will specifically focus on the structure and function of the Golgi.

Structure of the Golgi

The Golgi was named after an Italian researcher by the name of Camillo Golgi in the late 1800s. He discovered the Golgi during his exploration of the central nervous system by microscopy. (It is for this reason that we always write the name of the organelle with a capital G.)

Figure 04-23 showcases the common structural features of the Golgi and highlights many of the new terms you will need to know to discuss this organelle. The following are some important points to note:

• Each of the “pancakes” of the Golgi is called a cisterna (plural cisternae).
• The interior space of each cisterna is known as the lumen. You have seen this term before, as it is also used to identify the space inside the ER, called the ER lumen.
• The Golgi is considered to be a “polar organelle” in that cargo enters at one end (the cis face, for newly synthesized proteins) and exits at the other end (the trans face). As such, we identify each of the cisternae by their location within the stack:
  • The cis cisternae are the ones nearest the cis face, which receive vesicles from the ER.
  • The medial cisternae are in the middle of the stack. There can be more than one.
  • The trans cisterna is closer to the trans face of the Golgi stack.
  • The trans Golgi network (TGN) is the trans most cisterna of the sac. Often it is more convoluted, as vesicles will be budding and fusing at this site. We’ll hear more about the TGN later.

The structure of the Golgi apparatus is both surprisingly variable between different kingdoms/species and yet also very similar. For example, in all eukaryotes, the Golgi is made up of a series of flattened sacs that work together like an assembly line, moving cargo through them in order. However, the location and arrangement of those sacs can differ.

• In animals, the cisternae of the Golgi are large and are located centrally, near the nucleus (thus we say they have a perinuclear position). Usually, there is only one, or maybe two, Golgi in an animal cell.
• In plants, on the other hand, Golgi stacks are very small and tumble through the cytoplasm, using a combination of cytoplasmic currents and the actin cytoskeleton to help them move through the cell. There can be hundreds of these tiny Golgi stacks in a plant cell.
• In yeast and other fungi, The Golgi arrangement is quite variable. In some fungi, it is more “animallike,” while in others, it is more “plantlike.” In still others, it is different yet again. In the yeast Saccharomyces cerevisiae, the individual cisternae of the Golgi move around the cell independently of each other.

In this chapter, we will primarily focus on the features of the Golgi that are consistent across kingdoms, with only a few highlights of the unique characteristics specific to particular cell lineages.

While the cisternae do look like flattened sacs in cross section (Figure 04-23), there is more complexity to the structure. Vesicles are regularly budding and fusing with the margins of the cisternae as material is moved forward or backward within the Golgi stack. As such, if you were to look at a single Golgi cisterna from the top, it would look a bit more like a “paint splat” than a nice round pancake. This can be observed in the electron micrograph below (Figure 04-24).

Localization of Resident Proteins Can Be Quite Specific

Within the Golgi there is a net flow of newly synthesized proteins (i.e., the cargo) from cis to trans. In addition, each part of the organelle has specific proteins that are residents in that region. For example, there are specific proteins that are located in the cis cisterna of the Golgi that are required to remain there in order to carry out their function. The same is true for the enzymes that are found in the medial cisternae, the trans cisterna, and also for those that locate to the TGN. This arrangement can be visualized exceptionally well using immunolabeling and a technique known as electron tomography (Figure 04-25). Tomography is a specialized technique that allows us to create 3D models from the sample sections we normally use in an electron microscope. In each of the panels of Figure 04-25, we see an example of a 3D reconstruction of a Golgi stack with different resident enzymes that have been labeled using gold particles attached to an antibody. The colored dots (yellow in A and C; red in B) represent that gold particle label.

Just like we’ve seen with other organelles, the ability of the proteins of the Golgi to localize so specifically can be traced back to the order of amino acids found within their primary sequences. There are specific sequences that are used to allow proteins to become part of a COPI- or COPII-coated vesicle and other sequences that allow proteins to be retained as a resident in the cis, medial, or trans Golgi (instead of moving through the cisternae and out the other side). As we learn more about the Golgi, we find that the specific localization of individual resident proteins might be even more precise, not only living within a single cisterna but also located in a specific region of that cisterna (inner versus outer ring). While you are not expected to know the details about the specific composition of these targeting sequences, it is important that you recognize that the ability of these proteins to localize so precisely is based on their amino acid sequence and can be traced right back to the DNA code they were made from.

Golgi Dynamics: How to Get from One Cisterna to the Next

One of the biggest challenges that the Golgi faces is that some proteins are “transient” and must move through the Golgi (i.e., from cis to trans), whereas other proteins are “resident” and need to stay where they are inside a given cisterna. As If that weren’t challenging enough, vesicles constantly fuse and bud from the different cisternae, adding and removing proteins and lipids from the membrane. So how does the Golgi maintain its resident proteins in the proper location while allowing other cargo to be pushed along through the system? Scientists have been trying to solve this dilemma for almost as long as the detailed structure of the Golgi has been known.

Over the years, a number of ideas have been put forth to explain this. As we learn more, different ideas have gained and lost favor. Currently, the most widely discussed models for transport through the Golgi are called the cisternal maturation model and the vesicular transport model (sometimes called the stable cisternae model). See Video 04-07 for an excellent explanation of the two.

While the two models are present as separate possibilities, the current thinking is that the method by which proteins are moved and processed in the Golgi is probably more complex than any one model predicts (not really a surprise). The most widely accepted model right now is the cisternal maturation model (Figure 04-26), even though there is clear evidence that at least some things move via vesicular transport. The cisternal maturation model assumes that new cis cisternae are continually formed by the fusion of vesicles that are flowing to the cis Golgi network from the ER. The previous cis most cisterna is now a medial cisterna, as it has been pushed further along in the stack. In this model the trans cisterna is the oldest and it started life as a cis cisterna. The trans cisterna breaks up into tubules and vesicles to form the TGN.

According to the cisternal maturation model, the proteins that need to pass through the Golgi (like newly synthesized proteins) stay in place inside the cisternae and simply enjoy the ride as they move from cis to trans. On the other hand, since the different cisternae also have different functions, “becoming” a medial cisterna will require that the resident proteins that work in the cis cisternae be removed from the new medial cisternae and sent backward to the newly formed cis cisternae. This retrograde trafficking is thought to be achieved via COPI vesicles.

Protein Processing in the Golgi: Glycosylation

One of the most common posttranslational modifications of proteins is the addition of polysaccharides. This process is called glycosylation (Figure 04-27). The enzymes that carry out these reactions are located in the lumen of the ER and the Golgi apparatus and not in the cytosol. This means that the proteins being glycosylated are residents of one of the organelles of the endomembrane system, are bound for secretion, or are membrane proteins.

There are two major types of glycosylation: N-linked glycosylation and O-linked glycosylation. Most introductory cell biology textbooks focus primarily on N-linked glycosylation, as it is believed to be the more common of the two. The biggest difference between the two types of glycosylation is how and where the sugar is attached to the protein. In N-linked glycosylation, the sugars are added to an asparagine (which has the 1-letter code N) within a specific amino acid sequence, whereas in O-linked glycosylation, it is a serine or threonine that gets used to covalently link the sugars (specifically, an oxygen in the R group gets used, hence the name O-linked). Otherwise, the process is somewhat similar in each case.

Glycosylation begins in the ER. In the initial step of the process, a prefabricated oligosaccharide “tree” consisting of N-acetyl-glucosamine, several mannose sugars, and a few glucose residues is transferred from a lipid known as dolichol (Figure 04-27, Step 1). Like all other lipids, dolichol is assembled on the cytosolic side of the ER membrane and then flipped to the lumen (cisternal) side of the membrane by a flippase (see Chapter 2, Topic 2.2). After the tree is transferred to the protein, the dolichol is free to be reused by adding a new oligosaccharide tree to it for another round of glycosylation.

After the “tree” is added to the protein, the sugars can be trimmed in the ER before being packaged into vesicles and sent to the Golgi apparatus. It is inside the Golgi where most of the glycosylation work happens. Each glycosyltransferase mediates the formation of a single type of bond between specific sugars (i.e., glucose, galactose, mannose, etc.). Thus, sugars are added one by one as the protein moves through the Golgi, like an oligosaccharide assembly line. Different glycosyltransferases reside in the different cisternae, which means that a glycosyltransferase that resides in a later compartment will be unable to add its sugar if a step in an earlier compartment does not happen. The result is an array of possible oligosaccharides that can be built onto the proteins, which can then be used for specific functions later on.

Cell Wall Production in the Golgi Apparatus

Of the various eukaryotic kingdoms, several of them include cells that are commonly surrounded by a cell wall (specifically, plants, algae, and fungi). Animals do not have cell walls, and protists are extremely variable in whether they have cell walls, not to mention what those walls are made of, which makes them a topic for a whole course on their own. The cell walls of plants, algae, and fungi are made primarily of polysaccharides. Since the Golgi is the location of virtually all of the glycosyltransferases that create new covalent bonds using sugars, it has a major role to play in the production of cell walls.

Cell walls are made of complex, often highly branched sugar chains that create a gel matrix in which cellulose is embedded. We saw examples of this before, in Chapter 2 (see Figure 02-20). These complex polysaccharides are synthesized one sugar at a time as the molecules move from cis to trans inside the Golgi, in a way similar to what we just saw for glycoproteins. They are packaged into vesicles in the TGN and then shipped to the plasma membrane, where they are secreted and then incorporated into the existing plant cell wall. The most common cell wall compound made in the Golgi is pectin, which is a highly branched molecule that helps hold all of the parts together.

The cellulose is not created in the Golgi but instead uses a special protein structure at the plasma membrane known as a rosette.

The Trans Golgi Network (TGN) Is a Major Cargo Sorting Center for Secretion

Once the proteins arrive in the TGN, it means that they have been through all of their needed processing and are ready to be sent to the next place / final destination. The primary function of the TGN is to act as a sorting center. Cargo moving through the Golgi will end up at the TGN, and based on the structure of the protein and/or the carbohydrates attached to it, it will be sorted into one of two major routes:

1. The secretory pathway (a.k.a. exocytosis): This pathway is for proteins that are destined either to be excreted into the extracellular space or to become an integral membrane protein in the plasma membrane.
2. The lysosomal pathway: As its name states, the lysosomal pathway is used for proteins that are residents of the lysosome.

As a result of its role, the TGN is less “pancake-like” than the other cisternae of the Golgi. In electron microscopy, vesicles are often captured in a variety of states of budding, making the TGN somewhat bulbous (Figure 04-23). There is quite a lot of activity at the TGN, as it ensures that each protein gets packaged into the correct transport vesicle so that it can be sent off to the correct destination for its function.

Interestingly, despite the fact that we spent a whole topic examining vesicle formation using coat proteins, our understanding of how cargo gets packaged into vesicles at the TGN is vague. In some cases, the structures produced are too large to be packaged into standard coated vesicles. There have been debates as to whether the TGN is able to peel off from the rest of the Golgi stack and act as its own transport compartment. In addition, recent findings show that both COPI and COPII coats are able to produce larger tubules as well as smaller vesicles, though it is still unclear how often this happens. Additionally, there are other cases where there is no evidence for any known coat protein being involved. Since the trafficking of vesicles at the TGN is an essential cellular process, it is very difficult to study. There are still a great many unanswered questions in this field.

Topic 4.4: Pathways of the Endomembrane System and the Techniques to Study Them

After newly synthesized proteins have traveled from the ER to the Golgi, they hit a crossroads at the trans Golgi network (TGN), where their paths diverge, depending on destination and function. As such, this is a good time to step back and take a look at the bigger picture of the endomembrane system and how proteins and other cargo travel through it. Thus, in this last topic of the chapter, we will discuss each of the major pathways that cargo takes through the endomembrane system. As is our practice in this textbook, we will end by exploring the experimental techniques scientists use to study how cargo moves through the different pathways.

First, to remind you, we will be looking at three primary pathways through the endomembrane system. They were originally identified way back 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 through the endomembrane system on their way out of the cell.
2. The lysosomal pathway, which is the path that newly synthesized digestive enzymes will take to get to the lysosome, which is their final destination.
3. The endocytic pathway, which is the path inward from the cell exterior for proteins and molecules brought in by the cell. Material brought in via endocytosis will also likely end up in the lysosome, where the macromolecules are broken up into their basic building blocks to be reused in biosynthetic pathways.

The Secretory Pathway (a.k.a. Exocytosis)

As mentioned above, proteins that are destined to be sent out of the cell (or to live in the plasma membrane) have two different versions of the secretory pathway that they might take (Figure 04-28):

1. In regulated secretion, proteins are packaged into vesicles that are stored in the cell until they are secreted in response to a specific signal.
2. In constitutive secretion, vesicles continuously form and carry proteins from the Golgi to the cell surface. This is also sometimes called the default secretory pathway, as it requires no special targeting sequence (beyond the ER insertion sequence required to first enter the endomembrane system) for cargo to get secreted via this pathway.

With two pathways that both lead to the plasma membrane and out of the cell, why might a particular protein be secreted via one pathway or the other? Consider, for a moment, why one protein’s exit from the cell might need to be controlled and another one might not. Can you think of examples of proteins that might be better served by traveling through one pathway or the other? These are questions to keep in mind as we explore the different forms of secretion.

Constitutive Secretion

The constitutive exocytic pathway operates continually in all cells and supplies a continuous stream of vesicles containing lipids, proteins, and polysaccharides to the plasma membrane for release outside of the cell. When we think of “secretion,” this is often the pathway we are referring to. Because it exists in all cells, and because it appears to require no specific signal for cargo to enter this pathway, we generally consider this to be the default pathway through the endomembrane system. A protein that is able to enter the endomembrane system will contain an ER insertion sequence. However, with no other targeting sequences, it will eventually get secreted out of the cell. Examples of components that are secreted via the constitutive exocytic pathway are the following:

• extracellular matrix components (in animals),
• cell wall components (in plants and fungi), and
• antibodies in activated, antibody-producing B-cells.

Cells that are currently undergoing a great deal of constitutive secretion often can be identified in transmission electron microscopy (TEM) due to the large amount of ER in the cytosol (especially rough ER).

Depending on the cargo, it is thought that constitutive secretion may, or may not, require coat proteins. Clathrin-coated vesicles have been shown to be involved in some cases, but there are other cases where clathrin is most definitely not involved. As we said, sometimes COPI or COPII gets involved, though this seems to be mostly for large cargo (such as collagen and other large extracellular matrix components) that is far too big to fit into a clathrin-coated vesicle. In some cases, we have evidence that the vesicles form and that transport is happening, but we have no understanding of how the vesicles are forming. Thus, this is another area of active and ongoing research.

Regulated Secretion

Like the name says, the release of vesicles carrying cargo in the regulated secretory pathway requires a stimulus from either inside or outside of the cell before the final fusion of vesicles with the plasma membrane is allowed to take place. The vesicles form and then sit in the cytosol until they are ready. As a result, in TEM, a high concentration of vesicles in the cytosol is considered to be a marker of a cell that is involved in regulated secretion.

It is thought that vesicle formation in regulated secretion is aided by the proteins that use this pathway for export. There is evidence that the cargo proteins tend to “clump together” in acidic conditions (we call this aggregation, and the “clumps” are known as protein aggregates). This is helpful, as the Golgi apparatus tends to become more acidic from the cis to trans sides of the stack (from a pH of about 6.8 in the cis cisternae to as low as 6.3 in the trans cisternae). The result is that in the TGN, we find these large protein aggregates, which, if large enough, can then push up against the membrane and help promote curvature. Like the constitutive pathway, none of the known vesicle budding machinery seems to be involved, so these protein aggregates are likely extremely important to the formation of vesicles in regulated secretion.

The regulated secretory pathway is used for products that must be secreted very quickly, but only on demand. For example, insulin is produced by the beta cells of the pancreas and stored in dense core secretory granules. When blood sugar increases to a threshold level, insulin-containing secretory granules fuse with the plasma membrane, releasing insulin into the blood. Some other examples of regulated secretion are the release of neurotransmitters in the nervous system and the release of breast milk during infant feeding.

In some cases, regulated secretion allows for further processing of the protein after it leaves the Golgi but before it gets sent out of the cell. For example, many of the secreted digestive enzymes in our gut are further processed after being packaged into vesicles. Often this involves a section of the protein being removed, which activates it. Our previous example, insulin, is another protein that undergoes processing while it is in vesicles, waiting to be secreted.

The Lysosomal Pathway

The other major pathway for proteins to take from the TGN is what we call the lysosomal pathway. The lysosome is a specialized compartment that is the site of intracellular digestion. Lysosomes contain a mixture of some 40 different types of digestive enzymes, including those that degrade nucleic acids, proteins, carbohydrates, and lipids.

Like the Golgi, there is quite a lot of variation in the lysosome and endosome structures in different tissues and eukaryotic species. Plants are able to use their vacuole as a lysosome instead of having a separate compartment for it. In white blood cells that engulf bacteria as part of our immune response, the lysosomal enzymes are delivered to the large phagosome directly, using vesicles, rather than via the mechanism we will describe here.

It is also worth noting that lysosomes are but one of many different membrane-bound compartments that exist in this “post-Golgi” region (meaning the various destinations that the TGN may be sending cargo to after it leaves the Golgi). Lysosomes develop from another “post-Golgi” compartment, known as the endosome, using a mechanism that is similar to the cisternal maturation model of the Golgi. There are several different endosome-like compartments that are recognized, and discussed by scientists, based on their functions. The difference between the various compartments is fuzzy at times, as one compartment can “become” another compartment through the addition and removal of specific resident proteins. For example, we sometimes differentiate endosomes as “early endosomes,” which are not as acidic, and “late endosomes,” which are more acidic and have more characteristics of the lysosome that they will eventually become.

There are also recycling endosomes, multivesicular bodies, and a compartment called the endo-lysosome. While we don’t have time to go into all of the details of each of these compartments, this makes the point that we do our best to give you a set of well-defined “rules” to understand the processes we’re exploring. However, we cannot completely remove the complexity, as it underlies everything that we are going to talk about. This function of this part of the cell is a bit fuzzy, somewhat confusing, and also very likely species specific.

Targeting Proteins to the Lysosome

Like all of the other organelles we’ve explored, in order for a protein to be directed to the lysosome, it must have a specific sorting signal within the chemical structure of the protein that the cell can recognize. Lysosomal targeting is somewhat unique compared to the other targeting signals covered so far, as it is actually a carbohydrate signal that is attached to the protein via N-linked glycosylation (described previously in Figure 04-27). The carbohydrate signal used for lysosomal targeting is called mannose-6-phosphate, or M6P. As the name implies, the M6P signal is a six-carbon mannose sugar, which has a phosphate group attached at the 6-carbon. The M6P is part of a larger oligosaccharide that was added during N-linked glycosylation and is usually at the end of the chain that is farthest away from the protein it’s attached to. These modified mannose sugars can be identified by receptors in the TGN so that they get packaged into vesicles and sent to the endosome (Figure 04-29).

Here’s how it works:

1. N-linked glycosylation occurs as usual, with the oligosaccharide “tree” being added in the ER and trimmed.
2. In the cis Golgi, a phosphate is added to the sixth carbon of a specific mannose in the tree. The result of this is twofold:
   1. The M6P tag has been formed, which will be used later by the receptor.
   2. This also stops other glycosyltransferases in the Golgi from further modifying the sugar “tree.” That way the M6P tag isn’t masked or destroyed.
3. In the TGN, the M6P is exposed so that it can bind to the mannose-6-phosphate receptor (M6PR).
4. The protein-receptor complex gets packaged into clathrin-coated vesicles, and the vesicles are sent from the TGN to the endosome.
5. There, the lysosomal proteins are separated from their receptors (due to the increased acidity in the endosome).
6. The lysosomal proteins stay in the endosome, which will eventually mature into the lysosome, and the now-empty M6P receptors are recycled back to the trans Golgi network using a unique type of vesicle called the retromer.

Function of the Lysosome

The function of the lysosome is simple: its job is to break down cellular or extracellular material into its basic building blocks. These materials could include large polymers and other components brought in from the exterior via the endocytic pathway. Lysosomes are also involved in the breakdown of bacteria that are engulfed by the cell during a process known as phagocytosis as well as in the destruction of internal components that no longer function (such as a broken-down mitochondria). As a result, in some cases, the lysosome itself is the site of digestion, while in others (such as phagocytosis), the lysosome fuses with another compartment, which releases the digestive enzymes into the new compartment to aid in the digestion of larger structures.

The lysosome is a very acidic compartment. It usually has a pH in the range of 4–5. The acidic pH of the lysosome (and the endosomes they are derived from) is maintained by membrane-bound ATP-driven pumps that move protons into the lysosomal lumen. The lysosome has a single membrane that surrounds it. Additionally, all of the proteins in the lysosome are heavily glycosylated. This makes sense for two reasons:

• First, the targeting signal is a carbohydrate signal (M6P), so the only proteins that can get targeted to the lysosome are, by definition, glycosylated proteins.
• Second, carbohydrate coverings are often used as protection in extreme environments such as this one. For example, your stomach is also an extreme, highly acidic environment that has a very similar function to the lysosome, except on a much larger scale! The cells that make up the stomach lining are protected from harm by a thick layer of mucus. Mucus is simply made up of secreted complex carbohydrates. In the lysosome, instead of secreted carbohydrates, the proteins themselves are heavily glycosylated, which provides protection to the lysosomal membrane from the digestive enzymes within it.

One more thing to reiterate: in many organisms (plants, fungi, and many ciliated protists), there is a large vacuole but no obvious lysosomes. This is because vacuoles often perform the role of the lysosome in the cells that have them. There is quite a lot of variation in how this is arranged in organisms from different kingdoms, so we won’t say much about it, but it’s still important for you to know the relationship between the vacuole and the lysosome.

Lysosomes in TEM

Figure 04-30 shows an electron micrograph of three lysosomes in the cytosol of a ciliated unicellular protist. Lysosomes often contain what looks like strange bits and pieces. This material and the internal structure of the lysosome change over time because the contents are derived from the remains of cellular digestion. Sometimes the bits inside are very dark, and sometimes they’re not. This can make lysosomes a little bit difficult to identify in TEM, as their appearance changes depending on what they’re digesting.

The Endocytic Pathway (a.k.a. Endocytosis)

The final pathway through the endomembrane system is the endocytic pathway, also known as endocytosis. This pathway starts at the plasma membrane instead of the ER. In this pathway, material is brought in from the exterior of the cell to the endosome. From the endosome, it can go several places:

• Proteins and other structures that are destined for degradation will go to the lysosome to be digested.
• Some cell-surface receptors are recycled back out to the plasma membrane after releasing their cargo in the endosome.
• There is some evidence, especially in plants, that some endocytic cargo gets sent to the TGN, where it may be sorted and redirected to another site afterward.

You can trace all these pathways in Figure 04-15, found in Topic 4.2.

Types of Endocytosis

When we discuss the endocytic pathway, we are discussing three distinct forms of internalization (Figure 04-31):

5. Phagocytosis: the ingestion of large particles by cells. The particles are taken into phagosomes or food vacuoles. In multicellular organisms, this occurs primarily in specialized cells, like macrophages. However, aquatic single-celled protists, such as amoeba or paramecium, are also capable of this.
6. Pinocytosis: the ingestion of fluid, ions, and other small particulates via small vesicles <150 nm in diameter. This is carried out by all cells and is believed to be nonspecific.
7. Receptor-mediated endocytosis: the ingestion of specific molecules (e.g., cholesterol or iron) that are captured by cell-surface receptors to be packaged into vesicles.

We will look at each of these in turn.

8. Phagocytosis

In phagocytosis, bacteria or other large particles are brought into the cell using large membrane-bound compartments we call phagosomes. Phagocytosis generally occurs as a response to the activation of cell-surface receptors, which results in the opening of calcium channels. The increased internal calcium concentration then signals the actin cytoskeleton to rearrange. The phagosome is then formed. Phagocytic cells play an important role in the removal of foreign organisms (like bacteria) and in the scavenging of dead or damaged cells in the immune system of many animals. Many of our white blood cells are capable of phagocytosis. In single-celled protists, this is also a mechanism to ingest food.

Phagosomes are complicated organelles to produce, as they require a great deal of membrane, which needs to be pulled up and around the structure that is being engulfed. Actin is thought to be heavily involved in this process, as it is a major mechanism used by the cell to push the membrane outward (more on this in Chapter 6), but the mechanism is still under investigation. Once the phagosome has formed, a lysosome will fuse with it to release digestive enzymes into the compartment, which can then be activated as the compartment acidifies. Interestingly, the acidic phagosome is also a site of active immune warfare in our bodies. Some bacteria are known to use the phagosome to invade their host. They are able to block lysosomal digestion and remain functional inside the phagosome. The bacteria that cause tuberculosis, Legionnaire’s disease, and listeria all manipulate the phagosome to protect themselves from the immune system in humans and other mammals.

9. Pinocytosis

This type of endocytosis can also be thought of as constitutive endocytosis, because it is thought to occur all of the time. Pinocytosis is relatively nonselective in what it brings into the cell (water, small ions, and other nutrients). It is also one of the ways that the cell manages to maintain a constant size despite the continuous flow of vesicles to the plasma membrane via secretion. For example, macrophages ingest about 25% of their own volume in fluid each hour and an area of membrane equal to its entire surface every 30 minutes. This is offset by the outward flow of vesicles (including membrane, fluid, and cargo) through exocytosis.

Pinocytosis may or may not use clathrin-coated vesicles for transport. More commonly, a special structure called a caveola is used. Caveolae (plural) are formed using a different protein complex that includes a protein named caveolin. They’re not as well understood as clathrin or the COP coats, so we won’t discuss them further.

10. Receptor-Mediated Endocytosis

Receptor-mediated endocytosis is a much more selective process than pinocytosis. It’s triggered by the binding of macromolecules to receptor proteins at the cell surface. The increased concentration of bound molecules triggers the formation of a vesicle. Endocytic cargo may be concentrated up to a thousandfold by binding and accumulating on the cell surface before incorporation into vesicles. Cargo molecules bind to receptor molecules that then bind to the adaptor protein (adaptin) and coat protein (clathrin in this case) that result in their concentration in the area of the forming membrane bud that will give rise to the endocytic vesicle.

An excellent example of this is the internalization of cholesterol. As you know from its chemical structure, cholesterol is not very soluble in aqueous environments, such as our blood, and this makes it difficult to transport from our digestive tract (where it’s absorbed from our food) to the rest of the cells in our body. As such, the cell packages cholesterol into a structure known as a low-density lipoprotein (LDL). The LDL can travel through the bloodstream and get endocytosed by the cells that require it (Figure 04-32).

The following are the steps of receptor-mediated endocytosis of the LDL:

1. The LDL complex binds to receptors on the cell surface, which triggers the formation of clathrin-coated vesicles.
2. The vesicles then lose their coats and travel to and fuse with the endosome.
3. In the acidic environment of the endosome, the LDL separates from its receptor.
4. The LDL receptors are returned to the plasma membrane via transport vesicles. Cholesterol is released from LDL in the lysosome and available to use in membranes throughout the cell.

The Endosome Is Also a Sorting Center

The endosome is the second major sorting center in the endomembrane system (the first one being the TGN of the Golgi). It is the junction point between the lysosomal pathway and the endocytic pathway (Figure 04-32). This means that it must be able to differentiate between cargo that is headed to the lysosome and receptors that must be returned to either the TGN or the plasma membrane.

As if that were not complicated enough, endosomes receive receptors and cargo from the plasma membrane that may be headed to several different destinations:

• Many receptors are returned to the same plasma membrane domain from which they came, while their cargo is passed onto the lysosome. The LDL receptor is an example of this (shown in Figure 04-32).
• Some receptors are not recycled but instead sent to the lysosome to be degraded. This can be done with or without their cargo. Many growth hormones and receptors for irreversible developmental cues will follow this route.
• Finally, some receptors, along with their cargo, are transferred to another plasma membrane domain. This allows specific cargo to be passed from one side of the cell to another, in a process known as transcytosis. This is the process by which antibodies get passed from parent to child through the breast milk–producing cells when a child nurses.

Whether the cargo has the same fate as the receptor it rode in with or not is, in part, dependent on whether the acidic environment of the endosome causes cargo release. If the cargo is one that gets released by the acidic endosomal environment, then the cargo alone will end up in the lysosome, and the receptor is free to be recycled. Receptors that do not respond to low pH by releasing their cargo are more likely to travel with the cargo to the lysosome and get degraded.

Experimental Methods for Studying the Endomembrane System

As we have said many times in this chapter, the endomembrane system of a cell is essential, which means that the cell cannot survive without it. Essential cellular processes such as this create an interesting conundrum to researchers who wish to study them. One of the most common methods for studying a biological system is to disrupt it and study the effects. But when a system is essential, the cell dies if you disrupt it. Unsurprisingly, it is extremely difficult to learn much from a dead cell. Thus, scientists have had to get creative to find ways to study the secretory system in cells. Their methods have been very innovative and have been vital to our understanding of cell biology.

11. Visual Techniques—Microscopy

One of the most commonly used techniques these days to study how cargo moves through the endomembrane system is live-cell imaging of fluorescently labeled proteins. (Note: We have already spoken about visual techniques at length back in Chapter 1, so if you need a refresher, you may want to refer to the material found there.) We can combine live-cell imaging with other techniques described below to track how the system changes when mutations are made or blockages are temporarily induced.

As an example of the power of fluorescent microscopy to teach us about the endomembrane system, Video 04-08 shows the ER (green network) and the Golgi (purple blobs) in a plant leaf cell. In TEM we would see more detail of the structure, but since the samples must be dead and fixed, we cannot learn about the dynamics of these structures. In this live-cell video, we see the Golgi and ER both moving and changing shape dramatically throughout.

12. Biochemical Techniques

When researchers were first trying to figure out the endomembrane system, a common approach was literally to take it apart to investigate the job of each of these compartments in vitro (i.e., in a test tube). The biochemical techniques used were quite involved and complex. One common way to isolate different parts of the endomembrane system was to use a process called cell fractionation. In this process, the cells were literally put in a blender to break them up and then centrifuged at varying speeds, under different conditions, to separate rough ER from smooth ER from Golgi and so on. Researchers could then work with isolated membranes to see what each one was capable of on its own. In biology, isolating your “thing” of interest is a very common way of trying to understand function. Figure 04-33 shows an example of reconstructing an ER from isolated membranes in a test tube for an experiment.

13. The Development of Temperature-Sensitive Secretion (sec) Mutants in Yeast and the Rise of Mutational Analysis as the Primary Tool for Studying the Endomembrane System

One of the most common ways for cell biologists to study cellular function is to disrupt a protein’s ability to function (through mutation of its gene) and then see what processes are disrupted when that protein is absent. When studying essential processes like secretion and the endomembrane system, this can pose a challenge. Many of the proteins involved are so important that the organism cannot survive at all without it. As you can imagine, this creates difficulties for scientists who want to better understand how the proteins of secretion function. The discovery of temperature-sensitive mutations was a turning point in our understanding of many essential cellular processes—so much so that the combination of temperature-sensitive mutations and live-cell imaging techniques is a very large part of how endomembrane research is done today.

A temperature-sensitive mutation is one in which the protein amino acid sequence has changed due to the mutation, but it’s not so severe a change that the protein is nonfunctional. The protein is still able to fold more or less correctly. At certain temperatures (known as the permissive temperature, usually a few degrees lower), these proteins work just fine. They are stable and are capable of doing their job. However, if the temperature is changed even a few degrees (to what is known as the restrictive temperature), the protein becomes unstable, denatures, and is no longer able to maintain its function. As long as the cell is kept at the restrictive temperature, the protein will not regain its function. If left for long enough, the cell will die. If the cell is moved back to the permissive temperature, in time, the protein will renature, regain function, and thus the regular cellular processes will resume.

An excellent example of a temperature-sensitive mutation is that of the coloring of a Siamese cat (Figure 04-34). Siamese cats very famously have lighter beige hair on their bodies and darker brown hair on their extremities (paws, face, tail). The particular coat coloring of a Siamese cat is the result of a temperature-sensitive mutation in the tyrosinase enzyme, which is one of the many enzymes involved in producing the brown color pigment, melanin. The cat’s extremities are slightly cooler, so in these areas, the tyrosinase enzyme is functional, and the dark color is produced. Near the animal’s core, the temperature is a little bit higher, so the enzyme does not function, melanin is not produced, and the animal has albino fur.

One of the advantages of this system, from a cell biology perspective, is that a temperature-sensitive mutation is heritable, because a cell at a permissive temperature can survive long enough to divide and/or reproduce. This is important in experiments, as it is very challenging to generate these cell lines with mutations in specific genes. Even if one does regenerate the same mutated gene, there is the possibility that the same gene with a different mutation will not show the same effects, so being able to maintain a cell line with a specific mutation in an essential gene like the ones of the endomembrane system allows us to spend more time studying each mutation and thus learn more about cell biology as a whole.

For the endomembrane system, a great deal of work has been done on the budding yeast, Saccharomyces cerevisiae. There are multiple reasons why they were an ideal system in which to study secretion:

• They are single-celled Eukaryotes with a very short life cycle (~1–2 hours) that tolerate a wide range of temperatures. This makes them ideal for creating temperature-sensitive mutants and studying the effects.
• They have a small genome with very few genes compared to us (~12 million base pairs and ~6,200 genes). A simpler system is easier to study.

As such, a great deal of work has been done in budding yeast in order to study the endomembrane system and secretion. A great many temperature-sensitive mutants exist that have shed light on many different parts of the protein machinery that drives function. They are called the secretion, or Sec, mutants. One such example is Figure 04-35, where we see the difference in one of the mutants, called n-Sec1, as restrictive and permissive temperature.

Chapter Summary

The endomembrane system is a dynamic system involving many organelles, each with a unique job to do. Fittingly, the regulation of this system is equally dynamic and complex. In this chapter, we have discussed the roles of the ER, Golgi, endosomes, and lysosome and how resident and transient proteins are sent to or pass through these different compartments.

The ER is the entry point into the endomembrane, and proteins targeted anywhere in the endomembrane system contain an ER insertion sequence that is part of their primary sequence. Depending on the type and location of these ER targeting sequences, the protein can be either soluble or membrane bound. This starts their journey in the endomembrane system.

Proteins move to different organelles within the endomembrane system via a small membrane-bound vesicle. These vesicles provide specific transport based on the proteins contained in each vesicle and the targeting machinery on the surface.

The Golgi is the protein processing and sorting center. All proteins that enter into the endomembrane system pass through this hub. It is the primary site of glycosylation, and it is the primary site of production of cell walls in plants, fungi, and algae. Proteins can reach the cell exterior, be sent back to the ER, or be targeted to the endosome/lysosome from the Golgi.

Finally, a secondary sorting hub, known as the endosome, receives cargo from the cell surface and redirects it. Proteins and other molecules can be sorted and returned to the Golgi / cell surface or sent to the lysosome to be degraded.

This is a crucial system that is also quite dynamic, which makes it difficult to study. However, some pivotal experimentation as well as advances in technology have allowed scientists to gain significant insights into this highly intricate system. This chapter simply scratches the surface of the scientific knowledge on the endomembrane system and we hope that you find it as interesting as we do. If not, we hope that you have at least a deeper appreciation for the importance and complexity.

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