3.5 Cycling and re-use of membranes and traffic proteins
As already mentioned, a vesicle follows a cycle in which it gains its coat, is released from a donor membrane, moves to the target membrane, becomes uncoated, and fuses with the target membrane. Once a vesicle releases its contents by fusing with the target membrane, its components become part of the target membrane or of the lumen of the compartment bounded by the target membrane. The vesicular membrane that has fused with the target membrane needs to be retrieved to form new vesicles. Recovery of the membrane is achieved by the process of budding and membrane scission, i.e. formation of a new vesicle. For example, the recovery of synaptic vesicles involves a new round of budding and scission and involves the same set of coat components used for vesicle release.
Which coat protein, adaptor protein and GTPase would be involved in recovering synaptic vesicle membrane from the plasma membrane at an axon terminal?
Clathrin, AP2 and ARF (see Table 3).
In the synaptic vesicle recovery pathway, the adaptor protein AP2 binds directly to synaptotagmin (see Figure 26), a vesicle protein that may flag the stretch of membrane that contains other recently fused vesicular components. The formation of the clathrin coat and the recovery of the underlying membrane involves close interaction of the proteins with the lipids of the membrane, and the process is regulated by specific enzymes that act on phospholipids. After the coated vesicle buds from the plasma membrane, it moves to an early endosome (Figure 26).
After fusion of the coated vesicle with an early endosome, the retrieved synaptic vesicle proteins are ready to be reformed into new synaptic vesicles. To make a mature synaptic vesicle, it must first be replenished with neurotransmitters. Before this happens the interior of the vesicle is acidified by the activity of an ion pump, which uses ATP to pump protons across the membrane into the lumen of the vesicle. The acidic environment is essential in that it drives the uptake and storage of neurotransmitter by specific proteins present in the membrane of the vesicle.
Many small neurotransmitters are recovered after release into the synaptic cleft, in order to replenish the synaptic vesicles. Small neurotransmitters such as acetylcholine are regenerated by enzymes near the nerve terminal. For example, after its release, acetylcholine is rapidly cleaved into acetate and choline. Most of the choline is then taken up again into the nerve terminal, by a high affinity sodium-dependent choline uptake system, and used for resynthesis of acetylcholine. Thus, the depletion of neurotransmitters is prevented by their rapid resynthesis and packing into the small synaptic vesicles retrieved by the process of endocytosis. Vesicles can be recycled many times at the terminal by repeated exocytosis/endocytosis, which entails retrieval of vesicle proteins and refilling with neurotransmitters.
Membrane recycling also occurs between the Golgi cisternae and the ER. The fidelity of synthesised proteins is checked during the process of anterograde transport, and all proteins that are not properly modified or folded are returned to the ER in vesicles that also carry biologically dysfunctional proteins as well as ER-resident proteins that had escaped. An intermediate compartment between the ER and the Golgi is involved in the sorting process.
Which coat proteins are components of the vesicles moving in the retrograde and anterograde directions?
COPII coats the anterograde vesicles and COPI the retrograde vesicles (see Figure 16).
The process that regulates the exchange of a COPII to a COPI coat is still unknown. It seems that sequential coupling between COPII and COPI coats may be essential to coordinate bidirectional vesicular traffic between the ER and the pre-Golgi intermediate compartments.
COPI-coated vesicles also move proteins that were involved in anterograde transport back to the ER and between the Golgi cisternae, so that these components are continuously replenished. After reaching the Golgi complex and delivering their cargo, the vesicles depend for recycling on the retrieval signal and retrograde transport. For example, ER membrane proteins carry a dilysine motif (KKXX) that binds directly to the COPI coat. The recycling of the soluble ER proteins involves a C-terminal tetrapeptide (KDEL) that binds to a receptor (Section 3.2), which packages the protein into the COPI-coated vesicle. The affinity of the KDEL receptor varies depending on whether it is in the ER or the Golgi compartment (high in the Golgi; low in the ER), so the receptor binds the proteins in the Golgi and releases them in the ER.
How could a receptor have one affinity for its substrate in the ER and a different affinity in the Golgi apparatus?
The lumens of the two compartments have different pH values (Section 2.2), which affects the charge on amino acid side-chains and therefore protein folding and conformation.
The KDEL-containing proteins and KDEL receptors are not the only molecules that are recycled between ER and Golgi compartments; v- and t-SNAREs and many Golgi enzymes are also returned to their correct compartments.
There is growing evidence for more than one mechanism for protein recycling between the Golgi and the ER. Some proteins clearly have KDEL-like sequences whereas others, such as some glycosylation enzymes, lack such motifs but nevertheless find their way from the Golgi complex to the ER. The KDEL-receptor independent/COPI-independent pathway of recycling is much slower than the COPI-dependent one. This transport process, like the COPI-dependent pathway, requires the cytoskeleton and motor proteins, but it seems that these two mechanisms are completely unrelated, because no increase in COPI-independent recycling is observed in cells that have had their COPI-dependent pathway inhibited. Investigation of the two transport mechanisms has been greatly assisted by the use of toxins (Box 4).
Box 4 Transport of toxins
The orientation of intracellular trafficking (anterograde vs. retrograde) is often studied by use of protein toxins that enter the cell and are transported subsequently from the Golgi to the ER. For example, Pseudomonas exotoxin (PE), cholera toxin (CT) and Shiga-like toxins (verotoxins) enter the cell by endocytosis and travel to the trans Golgi network from where they are recycled to the ER. Upon arrival in the ER, the toxins translocate to the cytoplasm to exert their effects. Both PE and CT contain C-terminal KDEL or KDEL-like motifs, which facilitate their transport from the Golgi to the ER, through interaction with the KDEL receptor. These toxins provide conclusive evidence for the existence of COPI-mediated retrograde transport that recycles between the Golgi complex and the ER.
At present, biologists know something about the start and the end points of recycling pathways, but have less understanding of the routes of recycling or detailed molecular mechanisms. Moreover, there is very little knowledge on how the ER and the Golgi disassemble during mitosis. It is believed that an interruption in vesicular trafficking is likely to occur through a collapse of the Golgi back into the ER, increased fragmentation of ER membranes and inhibition of COPI vesicular fusion. This complex process of disassembly must be followed by reassembly, which involves activation of Rabs, TRAPPs, SNAREs and NSF-related factors and seems to be controlled by cell-cycle dependent kinases and phosphatases.
Yet again, lipids should not be forgotten. Lipid rafts also affect trafficking between the ER and Golgi compartments. Although the major metabolic pathways of cholesterol and sphingolipids have been elucidated, our understanding of the molecular mechanisms of lipid trafficking between subcellular organelles is, compared with our knowledge of protein trafficking, still elementary.