Mori, Kazutoshi

Kazutoshi Mori

Kyoto University

Walter, Peter

Peter Walter

University of California, San Francisco

For discoveries concerning the unfolded protein response — an intracellular quality-control system that detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures.

The 2014 Albert Lasker Basic Medical Research Award honors two scientists for their discoveries concerning the unfolded protein response, an intracellular quality-control system that detects harmful misfolded proteins in the endoplasmic reticulum and signals the nucleus to carry out corrective measures. Kazutoshi Mori (Kyoto University) and Peter Walter (University of California, San Francisco) identified core components of this process and unveiled unexpected aspects of its mechanism.

Approximately one-third of cellular proteins pass through the endoplasmic reticulum (ER), a netlike labyrinth of membrane-bound tubes and flattened sacs inside the cell. Work in the 1960s revealed that the ER sorts and transports proteins that are destined for export or the cell’s surface, and we now know that the ER allows cargo to pass only after applying stringent standards. In particular, proteins must assume correct three-dimensional shapes to perform their jobs, and the ER fosters this outcome. Furthermore, when unfolded proteins accumulate in this compartment, the cell bolsters the ER’s folding capacity. This phenomenon forms the linchpin of the unfolded protein response (UPR).

The first clues about this system’s existence emerged in the late 1970s, when researchers discovered that glucose starvation drives cells to boost production of particular proteins. Amy Lee (University of Southern California) reported in 1983 that the rise stems from an increase in the quantity of messenger RNA (mRNA) templates for these glucose-regulated proteins, or GRPs.

Three years later, Hugh Pelham (Medical Research Council, Cambridge) established that one of the GRPs, GRP78, resides in the ER and resembles a protein that prevents heat-damaged proteins from clumping. When glucose supplies drop, sugars that normally decorate some proteins are no longer available. Pelham proposed that the resulting sugar-deficient proteins stick together, perhaps because they misfold, and that GRP78, like its molecular relative, thwarts protein aggregation. Pelham also found that GRP78 was identical to another protein, BiP, that associates with partially assembled antibody molecules in the ER. In parallel, Mary-Jane Gething and Joseph Sambrook (University of Texas Southwestern Medical Center) showed that BiP attaches to misfolded forms of a different protein in the ER.

These findings hinted that BiP helps proteins fold; if true, manufacture of BiP in response to unfolded proteins would serve a clear benefit. The connection between glucose starvation and folding remained murky, however, and the model relied on that link. In 1988, Gething and Sambrook established that misfolded protein rather than sugar-adornment defects sends the alert to ramp up BiP output.

In 1989, yeast BiP surfaced. Its quantities also climb in response to unfolded ER proteins. Mori joined Gething and Sambrook’s lab as a postdoctoral fellow, and the group identified a short series of DNA letters that abuts the BiP gene. This sequence spurs molecular machinery to copy, or transcribe, the BiP DNA into mRNA when unfolded proteins accumulate in the ER; the sequence, when placed next to a different gene, similarly turns on its transcription.

Together, these observations suggested that cells must somehow monitor the abundance of unfolded proteins in the ER and transmit that information to the nucleus, which houses the genes. These events spark production of BiP and other proteins that promote folding, which reverse the problem. But no one knew how the nuclear equipment senses the ER environment.

Illustration of Ire1, ATF6 and PERK pathways

The complexity of Ire1

Independently, Mori, in Texas, and Walter, in San Francisco, placed the DNA stretch that Mori had uncovered next to a gene whose product makes a blue substance. When unfolded proteins accumulate in the ER and the engineered yeast cells send the usual signal to the nucleus, it stimulates not only typical UPR targets, but also the gene that turns the yeast blue. Yeast with defects in the UPR system would not change color, the researchers reasoned.

In 1993, the investigators used this scheme to isolate white yeast strains and thus tracked down the faulty genes whose normal versions presumably contribute to the UPR. One encodes a protein called Ire1.

Sequence analysis of Ire1 suggested that it is a kinase — an enzyme that adds phosphates to itself and/or other proteins. Additional work by Walter and Mori confirmed and extended this prediction. They found that Ire1 lies in the ER membrane with its kinase portion in the cytoplasm. In this orientation, the ER region could detect an unfolded protein signal and the other end could convey the message to cytoplasmic partners.

Mammalian kinases were well known to monitor environmental cues and, by adding phosphates to themselves or other molecules, trigger adaptive physiological changes. Perhaps, Mori and Walter reasoned, Ire1 would behave similarly.

To figure out how Ire1 delivers the unfolded-protein message, Walter and Mori (by then an independent investigator in Japan) set out to identify the presumptive courier that picks up the signal and carries it to the nucleus. They sought a protein that binds to the DNA sequences adjacent to UPR target genes and provokes transcription. The investigators captured the component they sought, a protein that previously had been named Hac1.

Their results, reported in 1996, contradicted expectation. In the simplest scenario, the theoretical protein to which Ire1 affixes a phosphate would be ready for action upon stimulation. Hac1, however, is not ready for anything; rather, it is manufactured only after the UPR alarm rings.

A crucial clue to explain this result came from the observation that the Hac1-encoding mRNA shrinks when unfolded proteins accumulate. Instead of adding a phosphate to another protein, Ire1 prompts removal of a chunk of Hac1’s mRNA. Additional work by Walter, which was confirmed and extended by Mori, established that HAC1 precursor mRNA contains an internal stretch of 252 genetic letters that is eliminated to supply the blueprint for active Hac1.

A canonical molecular machine splices sequences from precursor mRNAs and operates in the nucleus. The plot thickened when Walter showed that this apparatus does not act on HAC1 mRNA. Instead, he found, the severed HAC1 mRNA is stitched together by a cytoplasmic enzyme — tRNA ligase — that normally joins the two components of a different type of RNA, transfer RNA.

The search was now on for an enzyme that excises the middle piece of the HAC1 precursor mRNA. Inspired by a related protein’s behavior, Walter showed that the cytoplasmic segment of Ire1, which contains the kinase and an additional stretch of protein, could cut HAC1 precursor mRNA at the expected sites. Then he demonstrated that the splicing reaction could occur in the test tube with only two enzymes: Ire1 cleaves the HAC1 precursor mRNA at both splice junctions, and the transfer RNA ligase sews them together.

Mammalian systems unfold

As these details of the yeast UPR were materializing, researchers were struggling to gain traction in the mammalian system. In 1998, Mori unearthed a sequence that was common only to genes that fire up in response to unfolded ER proteins. This element rouses several UPR target genes, he found. Furthermore, a human protein called ATF6 binds to this DNA motif and activates adjacent genes.

Mori noticed that an overabundance of unfolded proteins incites conversion of full-length ATF6 to a smaller version; the large form dwells in the ER, whereas the trimmed one resides in the nucleus. This and other work suggested that excess unfolded proteins trigger release of a portion of ER membrane-bound ATF6. The liberated fragment travels to the nucleus and activates transcription of UPR target genes.

While Mori was discovering and elucidating ATF6’s role in the UPR, David Ron (New York University School of Medicine) and Randal Kaufman (University of Michigan Medical Center) found mammalian versions of Ire1, which share fundamental functional features with their yeast cousin. Three years later, Mori and Ron identified the human and worm versions of yeast Hac1, a protein known as XBP1.

In the meantime, near the beginning of 1999, David Ron and Ron Wek (Indiana University School of Medicine) had independently uncovered a third arm of the UPR, which depends on a protein called PERK. Like Ire1 and ATF6, PERK also lies across the ER membrane. Furthermore, its ER domain resembles that of Ire1. On the cytoplasmic side, a protein kinase segment of PERK adds phosphates to a particular protein, which then impedes translation of mRNAs. As a result, fewer proteins enter the ER, thus lightening the folding load.

Strength in numbers

In the last ten years, Walter, with UCSF colleague Robert Stroud, has peered more closely at Ire1 activation with X-ray crystallography. Previous work by Mori, Walter, and others had suggested that UPR induction causes Ire1 molecules to snuggle up in the membrane. By studying yeast Ire1, Walter and Stroud provided an atomic-level rationale for those results and illuminated details of the reaction.

In addition to providing assistance during protein folding, BiP attaches to Ire1 on the side that lies within the ER; when BiP falls off, naked Ire1 molecules pair up and create grooves that bind the unfolded proteins, Walter and Stroud suggest. Multiple Ire1 duos then congregate to form higher order structures; such association rearranges their cytoplasmic segments, positioning them so they can grab and then snip the HAC1/XBP1 mRNA, according to the model.

Researchers are still uncovering layers in the UPR. For example, Ire1 governs ER membrane synthesis and a system that shuttles recalcitrant unfolded proteins from the ER to a cellular incinerator. Even with these additional components, the unfolded protein burden sometimes surpasses the cell’s management capacity. That situation can trigger cell suicide, which obliterates unhealthy cells that might otherwise wreak havoc. Investigators are deciphering how the Ire1, ATF6, and PERK branches of the pathway help cells make life-and-death decisions.

Many scientists are now pursuing ways to harness the UPR for medical advantage. Certain forms of some inherited diseases that cause elevated cholesterol levels, cystic fibrosis, and retinitis pigmentosa produce abnormal proteins that do not fold properly and overwhelm the UPR.

Walter and Mori have unraveled a process with numerous unusual features. Their work has unlocked a multi-layered, highly choreographed system that lies at the heart of normal cellular function.

by Evelyn Strauss

Key publications of Kazutoshi Mori

Mori, K., Ma, W., Gething, M.J., and Sambrook, J.F. (1993). A transmembrane protein with a cdc2+/CDC28-related kinase activity is required for signaling from the ER to the nucleus. Cell. 74, 743-756.

Mori, K., Kawahara, T., Yoshida, H., Yanagi, H., and Yura, T. (1996). Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells. 1, 803-817.

Kawahara, T., Yanagi, H., Yura, T., and Mori, K. (1997). Endoplasmic reticulum stress-induced mRNA splicing permits synthesis of transcription factor Hac1p/Ern4p that activates the unfolded protein response. Mol. Biol. Cell. 8,1845-1862.

Haze, K., Yoshida, H., Yanagi, H., Yura, T., and Mori, K. (1999). Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol. Biol. Cell. 10, 3787-3799.

Yoshida, H., Matsui, T., Yamamoto, A., Okada, T., and Mori, K. (2001). XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 107, 881-891.

Sato, Y., Nadanaka, S., Okada, T., Okawa, K., and Mori, K. (2011). Luminal domain of ATF6 alone is sufficient for sensing endoplasmic reticulum stress and subsequent transport to the Golgi apparatus. Cell Struct. Funct. 36, 35-47.

Key publications of Peter Walter

Cox, J.S., Shamu, C.E., and Walter, P. (1993). Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase. Cell. 73,1197-1206.

Cox, J.S. and Walter, P. (1996). A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell. 87, 391-404.

Sidrauski, C. and Walter, P. (1997). The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell. 90, 1031-1039.

Gonzalez, T.N., Sidrauski, C., Dörfler, S., and Walter, P. (1999). Mechanism of non-spliceosomal mRNA splicing in the unfolded protein response pathway. EMBO J. 18, 3119-3132.

Korennykh, A.V., Egea, P.F., Korostelev, A.A., Finer-Moore, J., Zhang, C., Shokat, K.M., Stroud, R.M., and Walter, P. (2009). The unfolded protein response signals through high-order assembly of Ire1. Nature. 457, 687-693.

Gardner, B.M., Pincus, D., Gotthardt, K., Gallagher, C.M., and Walter, P. (2013). Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb. Perspect. Biol. 5, a013169.

Award presentation by Michael Brown

Award presentation by Michael BrownHenry Ford is credited with inventing the assembly line, but Mother Nature beat him by a billion years when she invented the endoplasmic reticulum, Mother Nature’s assembly line. In Ford’s assembly line parts are passed from one worker to another. At the end of the line is the inspector, who tests the product. Correctly assembled products are exported. Defective products are destroyed. If too many products are defective, the inspector sends a signal to the control room, where supervisors dispatch troubleshooters to correct the problem.

Ford never knew he was copying the endoplasmic reticulum, which I will abbreviate as ER. The ER is a network of membrane-bound tunnels within every nucleated cell from yeast to human. Like Ford’s assembly line, the ER makes products for export. Its products are proteins designed to function not in the controlled environment of the cell, but in the harsh environment outside. Some of the proteins are destined to reside on the cell surface, where they serve as receptors that respond to external signals. Other proteins are secreted to influence the behavior of other cells. Lasker laureate Günter Blobel showed that exported proteins are inserted into the ER during their translation. Once inside, they are bound by the factory workers of the ER, which are proteins called chaperones. Like Ford’s welders, the chaperones help the exported proteins to fold into their final three-dimensional structures. The ER assembly line must perform perfectly. Mistakes are not tolerated. Unlike in the auto industry, in biology there are no recalls.

Just like Ford’s line, nature’s assembly line has an inspector. When nature’s inspector finds defective proteins, it sends a signal to the cell’s control room, which is the nucleus. Just like a factory control room, the nucleus dispatches agents that enter the endoplasmic reticulum to correct the problem. The cell’s agents are additional chaperones. Their work is essential. If the problem is not corrected the cell’s secretory factory becomes congested and the cell dies. Even worse, if defective products escape the inspector and reach the circulation, they are recognized by the immune system as foreign, and this elicits an inflammatory response.

Peter Walter and Kazutoshi Mori, our two Basic Science Awardees, discovered the inspector of the endoplasmic reticulum. They showed how the inspector detects abnormal products and how it signals the nucleus. The mechanism is called the unfolded protein response. It is mechanistically unprecedented. It is conserved in all cells, and it is essential to life.

Mori was introduced to the protein folding problem as a postdoctoral fellow with Mary Jane Gething and Joe Sambrook at UT Southwestern Medical School in Dallas. I mention this because one of last year’s laureates, Thomas Südhof, also trained as a postdoctoral fellow at UT Southwestern. Not bad for a standalone medical school in a red state without a coast or a mountain. Mori’s co-recipient, Peter Walter, did not train in Dallas, but he did train in the United States. He trained with Günter Blobel at The Rockefeller University right here in New York, which has a coast but no mountain. I mention these facts because they show how the United States has been the endoplasmic reticulum of science. Like newly made proteins, nascent scientists are endowed by their genes with the potential for higher function. Like proteins, newborn scientists need chaperones to help them fold into the tough structure they will need for survival in the harsh external world. Until recently, the United States has had the best scientific assembly lines in the world. We imported the best raw material and turned out the best products. Now our scientific factories are threatened by short-sighted cutbacks in federal funding. There are people in today’s audience who are trying to reverse this downslide. Let us hope they get the support they need.

But I digress. Back to Kazutoshi Mori. He approached the unfolded protein response backward. He did not start with the first event — the sensor. Instead, he started with the last event — the signal that turns on the production of chaperones when the sensor is activated. From there Mori and Peter Walter worked backwards, eventually finding the sensor that detects the unfolded proteins and initiates the signaling.

Mori made his major discoveries after he returned to Japan, where he worked at Kyoto University. Walter remained in the United States at the University of California, San Francisco (which has a coast and a mountain). The two scientists worked independently in what appears to be a friendly competition, if such a thing exists. One of them would find a new link in the signaling cascade and the other would confirm the finding and use the information to discover the next link.

They both started by studying yeast, where powerful genetics allows a rapid dissection of biochemical processes. Soon they moved to animal cells, where the process was similar in principle but more complex in detail.

I will not burden you with the names of the proteins and the functions that Walter and Mori discovered. Those are listed in the fine articles that have been distributed to you today — by Evi Strauss in the official program, as well as in the pieces in Cell, Nature Medicine, and JCI. I will describe one aspect that is biologically unprecedented. When unfolded proteins accumulate in the ER, they bind to a sensor protein whose sensing domain projects into the ER lumen and whose active domain pokes through the ER membrane to function on the cytosolic side. When unfolded proteins bind to the sensor, they activate the cytosolic domain so that it becomes an active enzyme. The novel discovery is that the enzyme is a nuclease that excises a segment from a specific messenger RNA. Splicing activates the mRNA, allowing it to produce a transcription factor that enters the nucleus and activates transcription of the genes encoding chaperone proteins. The chaperones enter the ER and fix the folding problem. Before the Mori/Walter discoveries, mRNA splicing was believed to occur only in the nucleus. There was no precedent for the notion that such a reaction could take place on the surface of the ER, and that it could activate the translation of a specific protein. Mother Nature never wastes a good idea. It seems likely that other regulatory pathways must use a similar mechanism, but so far they remain undiscovered.

In humans, the unfolded protein response is essential in cells that secrete large amounts of proteins. Foremost are the antibody-secreting cells of the immune system whose secretions are crucial for our defense against bacteria and viruses. The liver also secretes lots of proteins that must be folded properly. A mutation in one secreted protein, alpha-1 antitrypsin, causes the protein to misfold and puts enormous stress on the unfolded protein response. Over time, the control room cannot keep up, and the patients develop liver failure.

The discoveries of Walter and Mori are among the most profound and original in all cell biology over the past several decades. They are worthy recipients of the 2014 Albert Lasker Basic Science Award.

Acceptance remarks

Interview with Kazutoshi Mori and Peter Walter

Video Credit: Susan Hadary