Sorting it out


It’s your friend’s birthday and you want to send them a gift. You wrap it carefully, write their address on the outside, stick on a stamp and put it in the mail. Then, as if by magic, it arrives at their house a couple of days later.

The magic happens in the postal service sorting office as mail workers recognise the stamp and the address, then send the package out with the delivery vans to the correct location. And almost exactly the same thing happens inside our cells on a much smaller scale, as they package up and send out parcels of proteins such as enzymes or hormones out into the body.

This secretory pathway, as it’s known, has been studied in great detail over the years. We now know that these secreted proteins are processed in a kind of cellular ‘factory’ known as the endoplasmic reticulum (ER) then sent to a structure called the Golgi body, where they are modified and packaged. The proteins are sent the right way thanks to molecular ‘stamps’ and ‘addresses’ – short regions within a secreted protein that mark it for export.

“Most proteins go through the endoplasmic reticulum and Golgi body for secretion – it’s a route we know very well,” explains CRG group leader Vivek Malhotra “But there are other secreted proteins that don’t make this journey, and we don’t know the ‘stamps’ and the ‘sorting offices’ that send them on their way.”

An unconventional journey

The story starts back in 2007, when Malhotra and his team noticed that yeast and slime mould cells secrete a protein called Acb1 when they are starving. But, strangely, Acb1 lacks any of the characteristic signals that should send it through the usual secretory pathway.

Instead it is gripped by special carrier proteins and taken to a temporary ‘sorting office’. This is the Compartment for Unconventional Protein Secretion (CUPS), which is built from components borrowed from the ER, Golgi body and small packages known as endosomes, and only appears under stressful conditions.

Malhotra and his team were curious about whether there were any other proteins that took this unorthodox route out of the cell, and decided to take a closer look at superoxide dismutase 1 (SOD1) – a protein that usually protects us by mopping up toxic chemicals in the body. Like Acb1, SOD1 doesn’t have the usual export ‘stamp’ that directs it through the ER and Golgi body, yet it is still secreted from cells.

SOD1 has been implicated in the neurodegenerative condition amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig’s or motor neuron disease). This is a rapidly progressing and incurable disease which kills nerve cells that trigger movement (motor neurons) and eventually leads to paralysis and death, and around a fifth of people with ALS have an inherited fault in the gene encoding the SOD1 protein. Researchers think that faulty SOD1 is secreted by neighbouring cells and taken up by motor neurons, destroying the precious nerve cells instead of protecting them.

Because of this crucial role for SOD1 in ALS, Malhotra and his team wanted to find out whether it also uses the CUPS route to get out of cells, and also to discover the identity of the biological ‘stamp’ that sends it there.

Starving and sorting

To keep things simple, the scientists started their search in yeast cells, which have very similar secretory pathways to human cells but are easier to grow and study in the lab. They noticed that when they grew the yeast under nutrient-rich conditions the cells secreted a little bit of SOD1. But when the cells were starved of nutrients, they exported nine times the amount.

Next, Malhotra and his team used genetic engineering to change certain molecular building blocks (amino acids) in SOD1, focusing on a region that is the same in both the yeast and the human versions of the protein. They discovered that a sequence of just two amino acids was enough to act as a ‘stamp’ to send the protein to the CUPS pathway. And, crucially, the same two amino acids are also found in the other unconventionally secreted protein Acb1, suggesting that this might be a universal signal for CUPS.

Finally, to see whether this same pathway might be at work during the development of ALS, the researchers tested healthy and ALS versions of SOD1 in human cells and found that it also is exported through the CUPS pathway when the cells are starved of nutrients.

Putting this all together, Malhotra believes that this work proves that both healthy and faulty versions of SOD1 are secreted from starving cells through the CUPS pathway, and that the little two amino-acid ‘stamp’ is enough to send them there. But there is still a mystery that needs to be solved.

“Many proteins have the same two amino-acid motif – in fact, it is extremely common,” he says. “We still need to find out how SOD1 and proteins like it are specifically recognised and sent to the CUPS, while other proteins are not.”

Malhotra thinks that the two-piece ‘stamp’ is normally hidden in proteins like SOD1 and Acb1 under normal conditions. But when something changes – for example, the protein is faulty or the cell is starving, which affects the shape of proteins – then it becomes exposed. Molecular ‘chaperones’ then step in to prevent any further unravelling, and instead send the protein off to the CUPS to be secreted out of the cell.

The identities of these chaperones and the exact ways in which they shuttle proteins into the CUPS are currently unknown, but Malhotra and his team are busy tracking them down. They are particularly interested in finding out what triggers harmful SOD1 secretion in ALS patients and – more importantly – working out if they can stop it.

“The discovery of this unconventional ‘stamp’ directing the secretion of SOD1 is very exciting,” he says. “At the moment there are no effective treatments for ALS, so I hope that our findings might provide the basis for the development of much-needed therapies in the future.”

Reference work

Cruz-Garcia D, Brouwers N, Duran JM, Mora G, Curwin AJ, Malhotra V.

“A diacidic motif determines unconventional secretion of wild-type and ALS-linked mutant SOD1.”

J Cell Biol, pii: jcb.201704056. doi: 10.1083/jcb.201704056. [Epub ahead of print] (2017).