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Preparing Proteins for Life on the Outside

Carolyn Ott
Biochemistry Department
University of California, San Francisco
August 2001

A protein is a chain of small molecules that have been strung together and folded or bent into a shape to perform a certain function. All proteins are made inside cells but many proteins work outside the cell. To get out, these proteins take a special route through the cell. Our laboratory studies how the folding of a protein at the earliest stage of its journey out of the cell can be controlled. We are investigating how changes in how proteins are shaped can allow the proteins to perform different functions. Failure to properly control the shape of proteins can lead to disease because the proteins cannot perform the jobs they need to.

In order to get out of the cell, newly made proteins pass through a series of membrane-enclosed compartments where folding occurs. We study how a protein gets into the first of these membrane compartments, the endoplasmic reticulum (ER), and how, once inside, the protein interacts with other proteins that help it to fold. We use many different model proteins to learn about different aspects of translocation, the process proteins use to get across the ER membrane. How a protein folds affects the job it is able to perform. A piece of wire can be folded to form a paper clip or a hook. A paper clip cannot be used to catch a fish nor can a hook be used to keep paper together. A protein, like a wire, can only perform the function it was shaped for.

A cell must be able to differentiate between proteins that should stay in the cell and proteins that need to be sent out of the cell. To facilitate differentiation, secretory proteins the cell wants to export have a tag at the beginning of the protein. Similar to a luggage tag, this protein tag, called a signal sequence, is "read" by the cell and the protein is sent to the appropriate compartment. Cell biologists have conventionally believed the only job of the signal sequence was to target newly made chains to the ER. Just as you might cut off the luggage tag after you receive your bags, the signal sequence is cut off once the protein enters the ER.

Proteins have different properties than the lipids that make up the wall of the membrane compartments. The lipids are oily molecules whereas proteins are similar to water. Just as oil and water do not mix, proteins cannot pass through the greasy membrane of the ER. Instead, they must pass through a channel in the membrane, called the translocon. In mammalian cells, proteins go to the translocon just after they have begun to be made. This works because the signal sequence is the first part of the protein being synthesized, so it is quickly recognized. This newly-made protein chain, along with the protein-making machinery are sent to the translocon. As a result, proper protein folding happens after the protein has left the cytoplasm. Proteins associated with the translocon are responsible for helping the protein fold and for cutting off the signal sequence.

Our lab recently found evidence that in addition to its role in targeting proteins to the ER, the signal sequence may also be involved in regulating how proteins fold. We found that putting a foreign signal sequence onto a protein can cause that protein to be folded differently. Because cells cannot change the signal sequence on a protein, we hypothesize that cells can achieve similar differences by switching the factors interacting with a signal sequence. By doing this, a cell could fold a protein a certain way to perform a specific function, or fold the same protein a different way to perform a different function. To demonstrate this we want to show the signal sequence can affect not only protein folding, but also protein function, the jobs the protein can perform.

The model secretory protein I am using to investigate the novel role of the signal sequence is called the TIGR protein. (TIGR stands for Trabecular meshwork Induced Glucocorticiod Response. This protein was found because it is synthesized in the trabecular meshwork cells of the eye when treated with steroids, specifically gluocorticoids.) Mutations in TIGR have been shown to cause glaucoma. Although I am looking mostly at how TIGR is made normally, what I learn may be used in the future to help discover how changes in TIGR cause disease. TIGR is a great model protein for my investigations because multiple functions for the protein are known. I am working with researchers in other fields to develop ways of measuring TIGR function. I will generate TIGR protein that has been synthesized using different signal sequences (which are cut off) and use the protein in different assays. I hope to demonstrate that changing the signal sequence can indeed change TIGR function, which would support the hypothesis that during translocation proteins can be made in different ways. These results would be significant because cell biologists have barely begun to appreciate the new role the signal sequence plays in protein folding.