Discovery Talk Questions and Answers: Unfolding the UPR

Peter Walter
Assessments created by Dr. Yi Liu

Questions

1. In his talk, Dr.Walter briefly introduced endoplasmic reticulum (ER). Which of the following statement(s) is true (select all that apply)?
   1. Proteins enter the ER in an unfolded state as they come out of the ribosome.
   2. Proteins fold in the ER
   3. Proteins assemble into multi-subunit complexes in ER
   4. ER is the first station which proteins enter as they move through the secretory pathway to the surface of the cell
   5. None of the above
2.  Which of the following statement(s) is true about the unfolded protein response (UPR)?
   1. UPR is initiated by the accumulation of unfolded (or misfolded) proteins.
   2. UPR regulates gene expression in the nucleus.
   3. UPR leads to apoptosis.
   4. UPR helps cells regulate their secretory capacity.
   5. None of the above
3. In the talk, Dr. Walter refers to “a small element in the promoters of the target genes of the response that can be (…) put in front of a reporter gene, and then we can isolate mutants in the cell where when we induce unfolded proteins in the ER that no longer induce the Unfolded Protein Response by this induction of this reporter gene”.

   How is this “small element” referred to in his 1996 paper?

   1. Endoplasmic reticulum (ER)
   2. Unfolded protein response (UPR)
   3. Transcription factor
   4. Unfolded protein response element (UPRE)
   5. None of the above
4. Using UPRE reporter system in yeast, Dr. Walter isolated Ire1 and HAC1 as two important components of the UPR.

   Which of the following statement(s) correctly describes their functions and relationships? (Select all that apply)

   1. Both Ire1 and HAC1 are ER based transmembrane proteins and they form a protein complex.
   2. Ire1 is a transmembrane kinase that senses the unfolded proteins and transmits the signal.
   3. HAC1 is a transcription factor downstream of Ire1 that binds to UPRE.
   4. None of the above
5. In his talk, Dr. Walter states that his group identified the second UPR pathway gene HAC1 using a reporter system in yeast. The process is described in detail in his 1996 cell paper.

   Describe how they identified HAC1 to be an essential component downstream of Ire1.

6. In his talk, Dr. Walter states that HAC1 was only found in cells when the UPR is induced (07:37).
   1. How was this demonstrated in the paper?
   2. List two explanations for the observed changes in HAC1 levels after induction.
   3. What simple experiment did Dr. Walter’s group do to distinguish these two possibilities and discuss the rational behind this approach?
   4. If Dr. Walter’s group had performed real-time PCR instead, would they still have been able to get the same result?
      1. Proteins enter the ER in an unfolded state as they come out of the ribosome.
      2. Proteins fold in the ER
      3. Proteins assemble into multi-subunit complexes in ER
      4. ER is the first station which proteins enter as they move through the secretory pathway to the surface of the cell
      5. e. None of the above
   5. Explain what makes them more stable then HAC1 protein translated from unspliced mRNA.
7. In the 1996 paper that described the product of spliced mRNA, the authors state that the HAC1 proteins translated from the sliced mRNA are more stable.

Answers

1. a,b,c,d.Video (1:11) “And the organelle that I’m going to tell you about is the endoplasmic reticulum, which is the first way station which proteins enter as they move through the secretory pathway to the surface of the cell, and end up being secreted or inserted in the plasma membrane. The endoplasmic reticulum then is a place where proteins enter the ER; they have to fold; they have to become assembled from multiple subunits; they become modified, maybe a carbohydrate is added; disulfide bonds are formed. And all of these reactions are important to produce properly functioning proteins. And cells that are specialized, that make a lot of proteins and secrete them, have to have a lot of endoplasmic reticulum in order to carry out these processes with fidelity and with appropriately abundant machinery.”
2. a, b and dVideo (4:06) “And it’s called the Unfolded Protein Response because it initiates with an accumulation of unfolded or misfolded proteins in the endoplasmic reticulum. And these proteins, they are unfolded because there isn’t enough capacity to fold them properly from the machinery. These proteins then create a signal that’s being transmitted across the membrane of the endoplasmic reticulum that ends up eventually in the nuclear compartment where it turns up a vast gene expression program that makes, basically, more endoplasmic reticulum folding capacity, secretory capacity, capacity to degrade proteins that cannot be folded properly and so on and so forth. So it brings cells back into a homeostatic state that allows the load of secretory proteins to be balanced with the machinery available to carry out their task.“

   p. 391, Introduction: “The unfolded protein response (UPR) pathway allows eukaryotic cells to respond to changing conditions in the endoplasmic reticulum (ER) by regulating the synthesis of ER–resident proteins. The accumulation of unfolded proteins in the ER leads to increased transcription of genes encoding ER–localized chaperones that assist in protein folding (Kozutsumi et al., 1988; Lee, 1987). The ER and nucleus are distinct membrane-bounded compartments of the cell. The unfolded protein signal, therefore, must be sensed in the lumen of the ER, be transferred across a membrane (either the ER or the inner nuclear membrane with which it is contiguous), and be received by the transcriptional apparatus in the nucleus, where it modulates gene expression (reviewed in Shamu et al., 1994; McMillan et al., 1994).”

3. d; Unfolded protein response element (UPRE)

   p. 391, Introduction: “A second known component of the pathway is the unfolded protein response element (UPRE), a 22 bp sequence present in the promoters of genes that are activated by UPR. When situated within a heterologous promoter, the UPRE is sufficient to activate transcription in response to the accumulation of unfolded proteins.”

4. b and cVideo (5:57): “And the nice thing is that the first gene, which we isolated this way, turns out to be IRE1 and it encodes a transmembrane kinase. So by virtue of it being a transmembrane kinase, it already told us that this may be the signal transduction device sitting in the ER membrane, figuring out in one end what’s going on there, and transmitting that information across the bilayer. Very nicely, the second gene we isolated turns out to be HAC1, and HAC1 encodes a transcription factor that binds to all these promoter elements. So we then have the transmembrane kinase, we have a transcription factor, and of course the way we are thinking about that is very much in analogy to other transmembrane kinases, like growth factor receptors in the plasma membrane of mammalian cells, that this thing is activated and functions by a process of oligomerization in the plane of the membrane where as unfolded proteins accumulate, these kinase molecules come together, they start bringing the kinases together on the other side of the membrane where they are now juxtaposed so they can trans-autophosphorylate each other and that somehow leads to phosphorylation cascade downstream that activates the transcription factor. But it turns out that nothing could be further from the truth.”
5. They used the ire1 deletion mutant yeast strain harboring UPRE driven His3 construct to screen a multicopy genomic library for plasmids. Since this UPRE driven His3 construct is the only functional copy of HIS3 in the cell, cells will not grow on plates lacking histidine unless a UPRE-binding transcription factor is introduced into the cell. During this screen, three genes were found to activate the UPRE: IRE1, SWI4 and HAC1.

   The authors then checked the UPR response in mutant strains where each individual gene was deleted (Δire1, Δswi4 and Δhac1). They found that both Δire1 and Δhac1 mutants were unable to induce the UPR, while swi4 showed no difference compared to WT cells, suggesting that Hac1 (like IRE1) is an essential component of the UPR.

   p. 392: “(…) we screened a multicopy genomic library to isolate genes that, when overexpressed in cells deleted for IRE1, turn on the UPR constitutively. The UPR can be monitored using reporter enzymes whose synthesis is placed under control of the UPRE. (…)

   p. 393: “(…) we tested the ability of cells deleted for either gene to up-regulate ER–resident protein genes in response to Tm treatment. As previously described, wild-type cells induced transcription of KAR2 mRNA (Figure 2, lanes 1 and 2), whereas the response was reduced to background levels in cells deleted for IRE1 (Figure 2, lanes 3 and 4; Cox et al., 1993; Mori et al., 1993). Interestingly, Δswi4 mutant cells were indistinguishable from wild-type cells (Figure 2, lanes 5 and 6), indicating that SWI4 is not required for the UPR pathway. In striking contrast, Δhac1 cells, like Δire1 cells, were unable to elicit the UPR (Figure 2, lanes 7 and 8), suggesting that Hac1p is an essential component of the UPR. Because of these results, we focused our studies on HAC1 and did not pursue further the effects that led to the identification of SWI4.”

6. a. 1. HAC1 localization by immunofluorescence microscopy experiment: With this experiment, Dr. Walter and his team tried to identify the route along which the UPR is propagated form ER to nucleus by checking the intracellular localization of Hac1 in uninduced cells and induced cells. Surprisingly, they found that HAC1 are localized in the induced cells but showed almost no signal in uninduced cells.p. 395: “Figure 4. Hac1p Is Detected Only When Unfolded Proteins Accumulate in the ER (A) HA-Hac1p localizes to the nucleus in Tm- treated cells. Δhac1 mutant cells (JC408) harboring the HA-tagged version of the HAC1 gene were grown either in the absence or presence of Tm and treated as described in Experimental Procedures. Cells were visualized by light microscopy (Phase), and DNA was stained with DAPI. HA-Hac1p was visualized by indirect immunofluorescence using monoclonal α-HA antibodies and FITC– labeled secondary antibodies. Both the untreated and Tm-induced cells were fixed and stained identically, and equal exposure times for each column are shown.”

   2. They then did a Western Blot on whole cell extracts to check the expression of HAC1 protein in uninduced and induced cells.

   p. 395: “(B) Hac1p induction is specific for ER– localized protein misfolding and requires functional Ire1p. Tm treatment of wild-type cells (JC408 harboring the plasmid pJC316) and Δire1 cells (JC417 harboring pJC316) was performed as in Figure 2A.”

   6. b. The two possible explanations are:

   1. HAC1 proteins are degraded when it is not needed.
   2. HAC1 proteins are only synthesized when the response is induced.

   p. 395: “The changes in HAC1p abundance upon induction of the UPR could be due to transcriptional or posttranscriptional events or both”

   Video (7:25): “And he was the first one, very simply, very naively, just started looking for the transcription factor in cells that are either induced for the response or that are not. And as you can see here, the transcription factor is only present in cells when the response is induced. So that tells us two things right then and there, right? It’s either degraded when its not needed or it is only synthesized when the response is induced.”

   6. c. They performed a northern blot to check the mRNA abundance of HAC1 under uninduced and induced conditions. If the HAC1 protein abundance change is due to posttranslational degradation, then the mRNA level shouldn’t change. If the HAC1 protein abundance change is due to induced synthesis, then there should be an increased mRNA level in induced cells.

   Video (7:56): “And to distinguish between these two possibilities what Jeff did is he just did a simple Northern blot analysis by which he asked, “Does the messenger RNA encoding this transcription factor change in its abundance when we induce unfolded proteins?” As you see here, the messenger RNA doesn’t really change much in abundance. But what you see, what Jeff discovered here, is that we now have a band of a different size that was completely unexpected. So, the simple Northern blot then led to the discovery that there is something happening to the messenger RNA and to make a long story short it turns out that this messenger RNA becomes spliced.”

   p. 395: “This prompted us to test by northern blot analysis weather the amount of HAC1 mRNA was altered when the UPR was induced.”

   6. d. Not likely. The authors found that HAC1 mRNA abundance didn’t change much in response to induction. Instead, the mRNA are sliced and shows a smaller band via Northern blot. In real-time PCR, the detection is based on a small probe and a primer set that target only one region of the mRNA. Unless they use different probe sets that target different regions of the mRNA, real-time PCR will detect the mRNA abundance difference, not the mRNA size difference.

7. The HAC1 protein translated from unspliced mRNA contains a different C-terminal than the protein translated from the spliced mRNA. This C-terminal contains multiple clusters of PEST sequences, which are known to destabilize proteins by targeting them into ubiquitin-dependent proteolysis pathways.p. 398: “If both HAC1u and HAC1i mRNA are indeed translated to a similar extent, then Hac1pu must be degraded signif- icantly faster than Hac1pi. Inspection of the Hac1p sequence reveals clues about the mechanism of Hac1p degradation; the C-terminal half of the protein (see Figure 3A, amino acids 119–230) contains multiple clusters of the amino acids Pro, Glu, Asp, Ser, and Thr, which overall constitute 47% of the amino acids that make up this domain. Regions rich in these amino acids, known as PEST sequences, have been shown to destabilize proteins by targeting them into ubiquitin-dependent proteolysis pathways.”

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