Intracellular transport
Intracellular transport

Start this free course now. Just create an account and sign in. Enrol and complete the course for a free statement of participation or digital badge if available.

Free course

Intracellular transport

3.2 Formation of clathrin and COP-coated vesicles

The structure of clathrin and clathrin-coated vesicles is known in some detail. Clathrin consists of a heavy chain of Mr 180,000 together with a light chain of Mr 35,000. Clathrin molecules successively assemble into a polyhedral, cage-like coat on the surface of the coated pit. The clathrin coat is made of sub-assemblies, each consisting of a three-pronged protein complex, a triskelion, each leg of which is made of one heavy and one light chain (Figure 17a). The triskelion forms a lattice-like network of hexagons and pentagons (Figure 17b), which attaches to the membrane via an adaptor protein (AP) complex. Adaptor proteins bind both to clathrin and to integral membrane proteins of the vesicle and stimulate its assembly. Much more importantly, by binding to the molecules in the membrane of the vesicle, adaptor proteins appear to be responsible for recognising the appropriate cargo molecules.

Figure 17 The clathrin triskelion (a) forms a skeleton of hexagons and pentagons around the coated vesicle (b). (c) Scanning electron micrograph of clathrin-coated vesicles.

Clathrin concentrates in specific areas of the plasma membrane, forming clathrin-coated membrane invaginations, called clathrin-coated pits. Cell surface receptors cluster in the pits, and then through a series of highly regulated steps the pits pinch off to form clathrin-coated vesicles (Figure 18). Although the detail and ordering of the process is not fully defined, the main steps are:

  • Recruitment of the G-protein ARF (ADP-ribosylation factor-binding protein), adaptor proteins and clathrin to defined sites on the plasma membrane;

  • Assembly of clathrin, formation of clathrin-coated pits and cargo recruitment, specified by adaptor proteins;

  • Budding and detachment of the nascent clathrin-coated vesicles.

Figure 18 Transmission electron micrographs demonstrating successive stages in the progression from a clathrin-coated pit to a clathrin-coated vesicle.

Several types of adaptor protein have been characterised (Table 3); their distribution suggests that they correspond to coated vesicles with different origins. For example, the AP2 adaptor, found on coated pits at the plasma membrane, characterises endocytic vesicles. The AP1 adaptor is found on coated pits of the Golgi and identifies vesicles that are targeted to endosomes. The role of AP3 is not yet fully understood. In addition to clathrin and APs, a number of other molecules, including dynamin and epsin, have been implicated in the formation of coated vesicles at the plasma membrane.

Within the past few years a novel family of proteins has been identified as molecules that also interact directly with ARF, clathrin and vesicle cargo. These proteins appear to be ARF-dependent clathrin adaptors that facilitate specific membrane trafficking events, such as cargo sorting and vesicle formation at the trans Golgi network. They are named GGA proteins (Golgi-localised, gamma-ear containing, ADP-ribosylation factor-binding proteins). GGA proteins are cytosolic monomeric proteins containing four distinct domains, one of which, the ‘hinge’ domain, contains one or more clathrin-binding sites (Figure 19).

Figure 19 GGA proteins have been shown to act as multifunctional adaptors at the trans Golgi network. They bind to ARF–GTP and also bind to sorting proteins (sortilin and mannose 6-phosphate receptor, M6PR), clathrin and possibly other molecules. GGA proteins have four distinct domains, labelled VHS, GAT, hinge and ear.

As mentioned above, the deformation and scission of the vesicle is energy-dependent, and a family of proteins called epsins appear to be involved in this process. Epsin-1 induces membrane curvature and promotes the polymerisation of clathrin. Another protein, AP180, appears to limit the vesicle size, and vesicle scission is mediated by another protein, dynamin, a GTPase of Mr 100,000 that collaborates with the coat proteins to induce budding of clathrin-coated vesicles. Dynamin self-assembles into rings and forms collars at the neck of invaginated coated pits. These collars constrict the neck of the coated pits, which are then severed by hydrolysis of GTP (Figure 20).

Although the mechanisms are broadly similar, different coat structures seem to be involved in different transport steps. Hence, COPI-coated vesicles shuttle molecules from exit sites on the cis Golgi complex towards the ER, while COPII-coated vesicles shuttle them from the ER towards the Golgi. This type of spatial organisation of transport maintains an asymmetric intracellular distribution of different proteins, such as enzymes, and permits the transport of newly synthesised proteins to the plasma membrane. For example, in the Golgi network, modifying enzymes are spread in a unique gradient-like distribution across several discontinuous membrane-bound compartments that collectively make up the Golgi stack; in the ER, enzymes mediating post-translational modifications of proteins either coexist in a continuous membrane or are segregated spatially into ER subregions.

Figure 20 Dynamin is recruited by vesicle-associated proteins and forms rings around the neck of the invagination. GTP hydrolysis leads to constriction of the dynamin ring followed by pinching off of the vesicle.

In contrast to clathrin-coated vesicles, which carry rather specific proteins, COP-coated vesicles undertake bulk flow from the Golgi to the ER and back, and the mechanism of recognising the protein cargo seems to be different. The cargo of the COPI-coated vesicles is selected by binding of the cargo molecules to specific membrane receptors. COPI-coated vesicles are involved in the ER retrieval pathway; thus, this pathway is often called the retrograde transport pathway. In contrast, the transport from the ER to the Golgi, achieved by movements of COPII-coated vesicles, is called anterograde.

Although the formation of clathrin-coated vesicles is similar in principle to that of COP-coated vesicles, there are some differences in the detail. Formation of COPII-coated vesicles requires the ordered assembly of the coat from many different cytoplasmic components (Table 3), but the formation of the prebudding complex as well as severance of the neck differs from the processes involved with clathrin-coated vesicles. Budding of COPII vesicles is initiated by the G protein Sar1, which recruits two further proteins, Sec23 and Sec24, to generate a pre-budding complex. The elongated COPII prebudding complex is in marked contrast to the open honeycomb-like clathrin-coated prebudding complex. Moreover, the architecture and folding of the COPII-coat components bears no resemblance to clathrin adaptor complexes. These structural differences between clathrin and COPII coats suggest that budding might be accomplished by distinct mechanisms within different intracellular compartments.

The mechanism by which cargo is recognised depends on both lumenal and transmembrane proteins of the vesicles as well as the receptors that reside in the Golgi complex. For example, COPI vesicles select proteins containing a dilysine motif near their C-terminus, in the form of -KKXX-COOH, where K is lysine and X is any amino acid residue.

After vesicle scission and transport to the target membrane, the delivery of the cargo occurs by vesicle fusion with the target membrane. The coat components would be an obstacle to membrane fusion, so they must be removed. Shedding of the coat is regulated by hydrolysis of GTP in ARF–GTP (Figure 21). Studies on transport in nerve terminals have shown that proteins including ARF, dynamin and some epsins, which are phosphorylated in resting nerve terminals, become coordinately dephosphorylated following nerve terminal stimulation. This dephosphorylation is believed to promote recycling of the components of the clathrin- and COP-coated vesicles (Figure 22).

Figure 21 Assembly of vesicles at the Golgi cisternae. ARF–GTP binds to the membrane of the cisternae and recruits coat proteins and vesicle-targeted proteins carrying soluble cargo molecules (other proteins are excluded from the vesicle). v-SNARE molecules, which are required for fusion with the target membrane, are also incorporated into the vesicle membrane. The vesicle buds off and moves towards the target membrane, where hydrolysis of GTP in ARF–GTP leads to depolymerisation of the coat.
Figure 22 Electron micrographs showing COPI- and COPII-coated vesicles.

Take your learning further371

Making the decision to study can be a big step, which is why you'll want a trusted University. The Open University has 50 years’ experience delivering flexible learning and 170,000 students are studying with us right now. Take a look at all Open University courses372.

If you are new to university level study, we offer two introductory routes to our qualifications. Find out Where to take your learning next?373 You could either choose to start with an Access courses374or an open box module, which allows you to count your previous learning towards an Open University qualification.

Not ready for University study then browse over 1000 free courses on OpenLearn375 and sign up to our newsletter376 to hear about new free courses as they are released.

Every year, thousands of students decide to study with The Open University. With over 120 qualifications, we’ve got the right course for you.

Request an Open University prospectus371