Why are vesicles coated?

In eukaryotic cells, precise and efficient intracellular transport of vesicular cargo, including proteins and lipids, to its required specific target location is an essential yet intricate process. The cytoplasmic proteins that coat vesicles play a major role in this, by facilitating the selection of cargo – through concentration (localized accumulation) and specification – and initiating the structural formation of the vesicle itself (Jackson et al., 2012). A coatomer is the name given to the protein complex that coats transport vesicles, and there are two distinct types: COPI and COPII, which differ in structure and are associated with different intracellular transport routes (Gomez-Navarro & Miller, 2016). Additionally, the clathrin (another structural protein) coat is functionally analogous to the COPI and II coatomers, but differs in clathrin’s triskelion structure and the involvement of adaptins (adaptor protein sub-units); it too is involved in different intracellular transport routes (Boehm & Bonifacino, 2001). The importance of these coats is evidenced by the vesicular trafficking processes they facilitate, as well as the pathology of the various genetic disorders that can result from disruption to the formation of these coat protein complexes, often caused by a mutation in the genes that code for one or more of the protein complex sub-units or in genes that code for the chaperone proteins involved in the coating and uncoating process. This can lead to a diverse range of diseases, from COPA Syndrome (Steiner et al., 2022) to Parkinsonism (Lewis, 2021).

Clathrin-coated vesicles

Clathrin-coated vesicles (CCVs), which were the first type to be identified, are involved in intracellular transport from the cell’s plasma membrane to the endosome, the Golgi, or another destination in the cell, making them key elements in the facilitation of receptor mediated endocytosis (Goldstein et al., 1985), although other transport routes are facilitated by CCVs as well (as discussed with respect to AP1 and AP3 later). Various features of the coat give the vesicle both the essential structural and cargo selection qualities needed to enable vesicle formation via budding and loading with the correct specific cargo required.

Cargo selection

The two-layered structure of the coat of CCVs enables the loading of specific cargo, allowing specialisation of the vesicles. The coat consists of clathrin as its main protein component, with each sub-unit of clathrin being made up of three large (or ‘heavy’) and three small (or ‘light’) chains, which associate together to form a structure known as a triskelion. These triskelions then assemble into a larger basket-like structure, forming coated pits on the cytosol-facing surface of the cell plasma membrane (Alberts, 2015). The other major coat component in clathrin-coated vesicles are clathrin adaptor proteins, also referred to as adaptins (McMahon & Mills, 2004). While the clathrin protein forms the outer cage layer of the vesicle, the adaptor proteins form the inner layer of the coat, and bind the outer clathrin layer to the plasma membrane. This traps various transmembrane proteins in doing so, some of which are cargo receptor proteins, which capture cargo molecules inside the newly forming vesicle. Depending on the set of transmembrane proteins trapped, and by extension the soluble proteins with which they interact, different cargo will accumulate and be packaged into each clathrin-coated transport vesicle. Since each adaptor protein type is specific to a different set of cargo receptor proteins, this allows for specificity (Alberts, 2015); for example, the AP1 adaptor complex is responsible for transport from the trans-Golgi network to endosomes (Touz, Kulakova & Nash, 2004), while the AP2 complex is key in endocytosis, since it can associate with the plasma membrane (Conner & Schmid, 2003). Meanwhile, AP3 is responsible for the trafficking of proteins from the trans-Golgi network to lysosomes (Gupta et al., 2006).

Vesicle formation

The vesicle coat is essential in creating a free vesicle structure from the parent plasma membrane. The assembly of the outer cage-like layer (the clathrin coat) of the clathrin-coated pit generates force, although this is not sufficient on its own to correctly shape and pinch off a new vesicle from the plasma membrane (Alberts, 2015). A GTPase, dynamin, is recruited, and enables the strangulation of the neck of the budding pit by other proteins, leading to two leaflets of the plasma membrane being fused, causing the vesicle to become sealed, pinched off and free (Cocucci, Gaudin & Kirchhausen, 2014). There are two mechanisms through which this can be done: either directly, by distorting the structure of the bilayer, and/or by altering the lipid composition of the bilayer through the involvement of lipid-modifying enzymes (Alberts, 2015). The involvement of this GTPase also allows for the rate at which pits are pinched off (and therefore the rate of vesicle formation) to be regulated (Alberts, 2015).

Coatomers (COPI and COPII complexes)

The role of the vesicle coat is just as vital in coatomer-covered vesicles, since COPI- and COPII-coated vesicles are functional analogues and evolutionarily homologous to CCVs (Boehm & Bonifacino, 2001), although they are most commonly involved in the mediation of transport from the ER and the Golgi, and while CCVs are primarily mediated by protein-protein interactions, GTP is the main driver of coatomer formation (Alberts, 2015).

COPI

COPI-coated vesicles play an essential role in maintaining cellular homeostasis, and thus the proper functioning of cells. The COPI coatomer surrounds vesicles that originate from the Golgi – these could either be destined to travel back to endoplasmic reticulum (ER) (known as retrograde trafficking, which is essential to the recycling of ER-resident proteins, such as chaperones) or to other parts of the secretory pathway (Alberts, 2015). It consists of a single complex, containing seven sub-units (Hughson, 2010). The binding of GTP causes a confirmational change in a protein called ARF-1, which subsequently induces the formation of the coat structure (Yu, Breitman & Goldberg, 2012). Unlike COPII, there are no separate inner and outer coat layers, just a single coatomer (Lee & Goldberg, 2010). Without COPI, ER-resident proteins would be unable to travel back to the ER from the Golgi, which in the case of chaperone proteins could prevent disulphide bond formation and a range of post-translational modifications occurring as normal, which would ultimately be disastrous for the cell.

COPII

In contrast to COPI, COPII is better characterised, with distinct inner and outer layers, and is essential in facilitating the formation of vesicles emanating from the ER (Peotter et al., 2019). The Sec12 protein, which is present in the ER membrane, activates Sar1, a cytosolic GTPase (Peotter et al., 2019) (and ARF homolog (Barlowe et al., 1994)). GTP-bound Sar1 then inserts itself into the membrane, where it preferentially binds to areas of membrane curvature, initiating bending of the ER membrane which leads to the assembly of the inner and outer coats (Alberts, 2015) (Peotter et al., 2019). Disassembly of the COPII coat can, at a later stage, be triggered by hydrolysis of the Sar1-GTP to GDP (Alberts, 2015).

Disease arising from disruption to vesicle coat formation

The importance of these vesicle coats is perhaps best highlighted by the diseases that can develop in the absence of correct coat formation. In mice, the loss of Sar1B (a specific isoform of the GTPase required for initiating membrane curvature and ultimately COPII-coated vesicle structure formation) results in rapid death (Lu & Kim, 2020), while in humans, loss of Sar1B results in a combination of two rare autosomal recessive disorders: Marinesco–Sjögren syndrome (which causes intellectual disability, childhood cataracts, muscle weakness and sparse hair) (Lu & Kim, 2020) and Chylomicron retention disease (Peotter et al., 2019). (Chylomicrons, which are large lipid-rich particles that transport dietary lipids from the intestine to other tissues, are unable to form correctly since proper COPII coat formation cannot be initiated due to a lack of Sar1 to insert itself into the ER membrane (Georges et al., 2011), leading to insufficient lipid absorption, stunted growth and cardiomyopathy (Peretti et al., 2010), as well as a reduction in the availability of fat-soluble vitamins, potentially leading to vitamin E deficiency (Aguglia et al., 2000).) COPA Syndrome, a recently discovered autoimmune syndrome, is caused by mutations in the genes encoding the alpha sub-unit of COPI, and can cause alveolar haemorrhage, renal and lung disease and arthritis (Vece et al., 2016). The requirement for proper regulation of vesicle function and location is especially true in the nervous system, with axons requiring long-range vesicular transport, and neurotransmission’s reliance on synaptic vesicles (Morfini et al., 2009). Interference in the formation of clathrin-coated vesicles has been associated with the onset of neurodegenerative disease, for example mutations in DNAJ proteins, a family of chaperones in the clathrin-coated vesicle life-cycle, have been identified in multiple Parkinsonism-type disorders (Roosen et al., 2019).

In conclusion, vesicular coating, whether by coatomers or clathrin, enables the formation of vesicles from plasma or organelle membranes, in addition to the vital regulation of vesicle function and directionality, making vesicle coats essential in facilitating intracellular transport and synaptic neurotransmission, and therefore in the survival of eukaryotic cells and organisms. This is shown by the potentially devastating impacts of genetic disorders which disrupt the genes involved in encoding proteins involved in coat formation.