4.2: Vesicle Transport

Learning Goals

• List the four stages of vesicle transport, and list the protein machinery involved at each step.
• Explain how vesicle coats facilitate cargo loading and vesicle budding.
• Discuss the importance of maintaining specificity in both docking and fusion of vesicles at their target compartments and how Rabs, tethers, and SNAREs facilitate this process.

Introduction

As we mentioned in the previous topic, the ER is the point of entry for all newly synthesized proteins that either reside in the endomembrane system or are destined to be secreted. In addition, once proteins enter the endomembrane system, they do not return to the cytosol. This poses a unique challenge for the cell in that the organelles of the endomembrane system are not physically connected to each other, and yet they must transfer cargo from one organelle to the next without allowing cargo to “escape” back to the cytosol. As such, vesicles are used to shuttle cargo around the cell.

Vesicles carrying cargo bud from one of the membrane-bound organelles of the endomembrane system (ER, Golgi, endosome, lysosome, etc.). They then move through the cytosol to fuse with the next organelle in the endomembrane system or the plasma membrane (Figure 04-15). In this topic, we will examine the details of how vesicles form, travel, and then fuse with the membrane at their target destination.

Figure 04-15: The movement of cargo through the endomembrane system. Each arrow indicates a vesicular transport path between those organelles. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

It’s important here for us to take a moment and point out that vesicle trafficking is extremely complex! There’s a lot we know about how vesicle trafficking works, but there’s also a lot that we don’t know. Vesicles are produced by all of the organelles of the endomembrane system. They all have specific cargoes and specific destinations. Some organelles (like the Golgi) are producing separate vesicles that will be sent to any number of different locations. The cargo is sometimes quite large, making it very difficult to put in a vesicle (collagen is a great example). And yet it, too, must get packaged properly and sent to its destination. For each new insight we gain about vesicle transport, we also find out that there are exceptions to what we thought were “the rules.” This situation can become overwhelmingly complex if we also consider all of the specialized cell and tissue types, differences between organisms, and even differences between biological kingdoms and how their endomembrane systems have evolved to address their unique needs.

Despite the complexity, in this section, we provide you with a framework to understand the parts of vesicle traffic that apply in most situations. This will hopefully help you learn what to look for when encountering a new trafficking scenario.

All of these pathways through the endomembrane system rely heavily on vesicles to transport their cargo from one stage in the pathway to the next. Since the three major pathways (see Figure 04-01 to remind you of what they are) are also always running in tandem, we begin to see why traffic through the endomembrane system is so complex.

Vesicle Traffic: The Basics

The basic principle of vesicle trafficking is that vesicles must take the correct cargo from the donor compartment and deliver it to the correct target compartment. This means that

1. 1. • the correct cargo must get into the correct vesicle, and
      • the correct vesicle must get sent to the correct destination.

A good analogy to consider here is that of an Amazon order (or any other online/mail order). When an online order is placed, we expect a couple of things to happen. The first one is that the product that was ordered will be properly packaged with the rest of the things that were ordered and prepared for shipping. This implies that no one else’s order will be included in this package. The second is that the package will then be delivered to the correct address, which is identified by the original order form. If you have ever had an online order that was shipped incorrectly, you have an understanding of the importance of doing this correctly the first time.

Vesicle trafficking works in a similar way. Instead of mailing addresses, targeting sequences are used. These sequences determine which type of vesicle the protein will enter so that it can be sent to the correct destination. The exception is when the cargo is destined to be secreted via the secretory pathway. Since secretion is considered the “default pathway” of the endomembrane system, proteins that are in the ER that don’t have other targeting sequences will get scooped up into vesicles in a nonspecific way and sent along on their journey. We’ll see more about the different pathways through the ER later in this chapter.

Vesicle traffic is controlled by the protein machinery that helps the vesicle form, travel, and fuse with the target membrane. It is this protein machinery that is the focus of this topic.

In all vesicle traffic, there are four main stages to the process (see Figure 04-16):

1. 1. 1. vesicle budding
      2. vesicle transport
      3. vesicle docking
      4. vesicle fusion

It is sometimes said that there is a fifth stage, which is resetting the system so that the next round of trafficking can happen. Repeated rounds of vesicle traffic require that the trafficking machinery gets recycled back to its original location so that new vesicles can be formed and targeted. We will focus primarily on the first four stages of this process and only mention system reset briefly at the end of this topic.

Figure 04-16: Stages of vesicle budding, transport, docking, and fusion. (1) Budding: Membranes deform toward the cytosol and pinch off, becoming free floating vesicles. (2) Transport: These vesicles are carried toward the target location, often using the cytoskeleton. (3) Docking: Vesicles are brought closer to the target membrane. (4) Fusion: The vesicle fuses with the target membrane, and any soluble components are released into the lumen of the acceptor (target) compartment. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Each of these stages must be carried out correctly. There is no margin for error in vesicle traffic due to the potentially destructive nature of the cargo that is being transported. Remember that proper function of the endomembrane system is absolutely essential to survival, which means that errors may very well kill the cell. In this material, we will focus on budding, docking, and fusion. Transport of vesicles is carried out using a combination of the cytoskeleton (which will be discussed in Chapter 6) and sometimes short-range diffusion (which we are not going to discuss further).

Step 1. Vesicle Formation and Cargo Selection (a.k.a. Budding)

It is important to understand that membranes do not curve on their own. Energy input is required to change the shape of a membrane. As such, it is thought that virtually all vesicles require some kind of protein machinery to help them form. However, the exact protein machinery used in the formation of many types of vesicles has yet to be discovered. We still have a great deal to learn about vesicle traffic despite over 100 years of research on the endomembrane system.

While there is a variety of protein machinery used to help promote the formation of vesicles, we can identify specific elements that are very common when looking at different ways that vesicles can form (Figure 04-17). All of the vesicles that we are going to study require the following:

• Cargo receptor: Proteins destined for particular target organelles must be collected together in one spot so that a vesicle can be formed around them. This is accomplished by binding of the proteins to receptors in the membrane that recognize them. This helps create the specificity required for protein transport. From our online order analogy, the cargo receptor is what picks up the merchandise and puts it in the correct box and also helps ensure that no other merchandise ends up in the box by accident.
• Adaptor protein: The adaptor protein acts as a bridge between the cargo receptor and the coat protein. Usually there are more adaptors than coat proteins, so this allows for another level of specificity. Different adaptors can bind to different receptors but still form a vesicle using the same coat proteins.
• Coat protein: The coat protein provides the structure to support the bending membrane while the vesicle is being formed. There are many components in a vesicle that help determine the direction of transport, but specific coats are generally associated with particular pathways and directions of transport. We’ll see examples of this as we move through this topic.
• GTPase: A GTPase is a protein that binds GTP and hydrolyzes it. The energy released when the GTP is hydrolyzed can be used at any part of budding, though it appears to be primarily associated with the release of the vesicle coat after its job is done.

It’s also important to know that the lipid composition at the site of vesicle budding is just as important as the protein machinery. Membrane lipids found in ER exit sites, and other regions from which vesicles bud, are important in helping to assemble protein machinery, allowing the membranes to curve, and even as identity markers. So even though we will not discuss the lipids much in this chapter, you should know that the lipids are equally important to proper formation and function of vesicles.

Figure 04-17: The protein machinery required for vesicle budding. A coat complex containing a coat protein, adaptor protein, and a small GTPase help stabilize the forming bud. Cargo is loaded into the forming vesicle. Some molecules are brought into the vesicle by bulk flow, while others are specifically recruited via interactions with a receptor or the coat adaptor. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

Steps in the Formation of a Vesicle

1. Cargo proteins are held in place by transmembrane cargo receptor proteins.
2. An adaptor protein is a protein that binds to the cytosolic side of the receptor as well as a cargo receptor protein.
3. A small GTP-binding protein (called a GTPase) acts as a regulatory unit to determine exactly when the adaptor and coat proteins are allowed to bind to the receptor. It is in its active form when bound to GTP. Over time, the GTP gets hydrolyzed to GDP, which inactivates it, and the GTPase falls off the membrane.
4. Proteins involved in flipping lipids across the membrane (related to the flippases and scramblases of Chapter 2) initiate the curvature, which is the start of a vesicle bud.
5. Coat proteins assemble into a lattice or network on the cytosolic side of the forming bud to help stabilize and extend this curvature (budding) until a vesicle is formed.
6. To release the vesicle, the forming vesicle bud needs to be cut off from the donor membrane (a process known as scission). This action may be part of the function of the coat protein, or it may be done by a separate protein.

Once the vesicle has budded off, the coat is usually removed, the vesicle can be transported to the target organelle, and the naked vesicle fuses with its target membrane.

An Introduction to the Vesicle Coat Proteins

All coat proteins have two primary functions:

1. To stabilize membrane curvature in the forming bud
2. To capture cargo molecules for transport

There are at least six types of vesicles where the vesicle-forming machinery is currently known and many more types of vesicles where we are still unsure how they are formed. Of the six known types, this textbook explores three of them in more detail, as they bear the most similarities to each other and are the most well characterized. More specifically, we will be looking at the three major classes of coat proteins and their role in vesicle formation. The three types of coat proteins we will study are COPI, COPII, and clathrin. Each of these types of coats tends to be used in different locations in the endomembrane system and/or for different purposes. They are also considered to be the “canonical coats,” probably because they were discovered first but also because they appear to be the most widely used by the cell.

1. Clathrin coats were the first coat proteins to be discovered and as such are the ones that we know the most about. They are most commonly used between the trans Golgi network (TGN) and endosomes, lysosomes, and the plasma membrane. However, even in these sites, they aren’t always used. Clathrin coats have been shown to be important in specific kinds of traffic:
   • carrying newly synthesized lysosomal enzymes from the TGN,
   • bringing in molecules from the extracellular space via receptor-mediated endocytosis (and possibly some other endocytosis as well),
   • recycling unnecessary membranes from secretory vesicles as they mature, and
   • sending proteins out of the cell using the secretory pathway in highly regulated circumstances (like in neurons, where vesicles full of neurotransmitters are sent down to the axon terminus).
2. COPI coats tend to be used for retrograde traffic, which means traffic that goes “backward” through the endomembrane system. Specifically, COPI coats are used for either Golgi-to-ER traffic or moving cargo backward through the cisternae of the Golgi. Some evidence suggests they can also be in other areas, but only in very specific situations. COP stands for “coatamer protein.”
3. COPII coats are most commonly used for anterograde traffic, which means forward traffic. Specifically, they’re used for ER-to-Golgi traffic. They also have the ability to make vesicles of different sizes and shapes, unlike some of the other coat proteins, so they are used for the secretion of large molecules (like collagen, for example). As a result, COPII can also be found at the TGN, but they are not as common as clathrin. COPI and COPII coats were named in the order in which they were discovered.

In a nutshell, clathrin is primarily used in vesicles originating from the TGN and from the cell surface. COPI and COPII are mostly used as the coat proteins for vesicles originating from the ER and within the Golgi. Table 04-01 summarizes some of the structural details of these three coats as they relate to the coat components described in Figure 04-17.

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

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.

Figure 04-18: Cryo-electron microscopy of clathrin coat. This coat is made of 36 triskelions and is approximately 90 nm in diameter. A single triskelion is highlighted in blue. “Clathrin cage viewed by cryoelectron microscopy” by Mazuraan is shared under a CC BY-SA 4.0 International license.

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.

Figure 04-19: Molecular structure of the different coat proteins. (A) Molecular models from Noble and Stagg (2015). (B) Schematic coat proteins overlaying a sketched cage structure from Harrison and Kirchhausen (2010). Average diameters for all vesicle coats shown are roughly similar, ranging from 60 nm (COPI) and 90 nm (clathrin), with COPII somewhere in the middle. Panel (A) is from Noble, A. J., & Stagg, S. M. (2015). COPI gets a fancy new coat: An interconnected scaffolding of proteins bends the membrane to form vesicles. Science, Volume 349, Issue 6244, July 2015, Pages 142–143. www.jstor.org/stable/24748469. Reprinted with permission from AAAS. Please do not redistribute without permission of the rights holder. Panel (B) is included under fair use as described in the CBPFUOER. Please do not redistribute without permission of the rights holder.

Video 04-05: COPII vesicle formation. A host of molecular machinery comes together to form the vesicle as well as recruit the cargo into the forming bud.

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.

Figure 04-20: Traffic between the ER and Golgi is managed by COPI- and COPII-coated vesicles. Proteins bound for the Golgi are packaged into COPII-coated vesicles. On occasion, ER resident proteins also get trafficked to the Golgi, where they are recognized by the KDEL amino acid sequence and packaged into COPI vesicles that return to the ER. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

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.

Figure 04-21: Docking and fusion. Tethers bind to Rabs to help bring the vesicle close to the target membrane (docking). When close enough, SNARE proteins interact to fuse the membranes together. This image was created by Heather Ng-Cornish and is shared under a CC BY-SA 4.0 license.

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.

Figure 04-22: The mechanism of neurotransmitter release at the synapse. The four SNARE coils can be observed, zippered together as part of the core SNARE complex. Three proteins are a part of the SNARE complex: synaptobrevin (a v-SNARE), syntaxin, and SNAP-25 (both of which are t-SNAREs). Note that SNAP-25 has two SNARE coils. The rest of the protein machinery here is specific to neurotransmitter release and not necessarily present at other vesicle fusion sites. “Molecular machinery driving vesicle fusion in neuromediator release” by Danko Dimchev Georgiev, M.D. is shared under an unported CC-BY-SA 3.0 license.

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.

Video 04-06: From neuronal communication to SNARE recycle. Vesicle fusion requires the interaction of SNARE proteins. These coil together once they interact to bring the vesicle close to the target membrane. SNARE complexes are specific between donor compartment and target membrane. At the end, the SNAREs are uncoiled, and the v-SNARE is sent back to its original compartment so that it can participate in further rounds of fusion.