PAIRing up cargo proteins

To sense their environment and respond to it, cells secrete a variety of proteins or display them on their membranes. To mature, such proteins must travel through the secretory pathway, the entry point of which is the endoplasmic reticulum (ER). To exit the ER proteins are packaged as cargo in lipid coated vesicles. For this to occur efficiently many cargos require the help of dedicated cargo receptors. Our study takes the first systematic approach to map all cargos that rely on each of the known yeast cargo receptors and lays the foundation for the first cellular traffickome.

HFSP Career Development Award holder Maya Schuldiner and colleagues
authored on Mon, 04 June 2012

Imagine sorting a pile of thousands of unmatched socks into pairs. Now imagine those pairs are microscopic. That begins to approach the challenge faced by Dr. Maya Schuldiner of the Molecular Genetics Department and her colleagues and students when they set out to identify pairs of proteins – proteins that get transported out of a cellular organelle coupled with the proteins that escort them to their rides. But Schuldiner and her team had some help: They adapted the robotic equipment in her lab to develop a system they call PAIRS, which prepares and sifts through thousands of samples to identify matches.

Scientists have been searching for such pairs for the past 20 years with only partial success. But the issue is vital: The proteins that escorts match up with are hormones, growth factors and various other signaling molecules that are produced for export to other cells or organs, and their activities are implicated in  many diseases, from autoimmune syndromes to cancer. A better understanding of the escorts’ functions could point to possible drug targets for treating these diseases.

The first way station for all proteins that either need to make it out of the cell or end up displayed on the cell’s outer surface is the endoplasmic reticulum (ER) – a maze-like organelle composed of folded internal membranes. The proteins entering the ER must get folded into shape as well as undergoing quality control testing before exiting the maze. But leaving for the next way station – the Golgi apparatus – for final sorting and routing, is a much more complex affair than entering. The now functional, folded protein must be enclosed in a bubble of membrane that buds off of the ER, creating a vesicle – a sort of private taxi that delivers its passenger to the Golgi apparatus without letting it contact the cell’s interior. This is where the escorts come into play. They sort and package the proteins – ensuring that only mature proteins leave the ER and in the right vesicles.

Until now, identifying escorts and finding their matches has been something like trying to sort through all those socks by hand, one at a time. That is why only 10 escorts had been identified, and these were matched to only a handful of proteins. And that, says Schuldiner, is not enough to begin to understand the rules of protein trafficking.

To remedy the situation, she and her team, including research student Yonatan Herzig and Dr. Yael Elbaz, together with Prof. Sean Munro and Hayley Sharpe of MRC Laboratory of Molecular Biology, Cambridge, UK, decided a more systematic approach was called for. In the PAIRS method they developed, a lab robot prepared and cultured yeast cells. The samples – grown in rows of tiny wells – each contained a yeast strain that had been genetically engineered so that one of 400 different proteins would glow fluorescent green, while one of the ten known escorts was rendered inactive. Producing all the possible combinations – just to find matches for the known escorts – required 4,000 different samples. Then, a second robot automatically scanned images of the cells, looking for a tell-tale green glow that showed that a particular protein was building up in the cell rather than being exported – a sure sign of a match.

For each escort, new passenger proteins were identified. At that point, says Schuldiner, the team could begin to formulate some rules about the ER transport system. For instance, the scientists found that the escorts mainly worked with relatively small protein groups, each of which used just one kind of escort. In some cases, the group of proteins using a particular escort had similar functions; in others, a shared chemical “password” gave them access.

Even more interesting was the one escort protein that seemed to be an exception to the rule: The scientists noted that Erv14 pairs up with an unusually large number of proteins that apparently have nothing in common. After a series of experiments ruled out all sorts of possible factors, the team hit upon the one thing that they all share – an extra-long domain that is required for them to be displayed on the outer plasma membrane of the cell.

Because versions of this escort are found in everything from yeast to fruit flies to mammals, the same rule should apply to human Erv14 and the proteins that it pairs with. One of those using the Erv14 escort is EGF Receptor protein, a protein required for proper embryonic development that also plays a well-studied role in cancer growth. A better understanding of the requirements for EGF receptor trafficking is essential for developing models of development as well as tumor progression.

In addition to the matches the researchers managed to identify, there were many proteins that didn’t pair with one of the known escorts. Do these forgo the help or do they use other, as-yet-undiscovered escorts? Schuldiner and her team plan to continue investigating. Their eventual goal is to produce a “traffickome” that will map out the transportation systems for all the proteins in the cell.

Text by Judith Halper of the Hamachon Journal

Reference

A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by erv14.  Herzig Y, Sharpe HJ, Elbaz Y, Munro S, Schuldiner M. PLoS Biol. 2012 May;10(5):e1001329. Epub 2012 May 22.

Pubmed link