Final Exam

1. Involved in nuclear import/export 2. When bound to GTP, binds import receptor, and releases cargo into nucleus 3. Plays a role in targeting vesicles to correct membrane 4. When GTP is hydrolyzed releases vesicle and effector

1. Ran 2. Ran 3. Rab 4. Rab

Select all CORRECT statements about transcription:

1. Transcription is carried out by RNA polymerase 2. In eukaryotic cells, following transcription, RNA is capped, spliced, and polyadenylated 3. Transcription results in the production of RNA 4.During transcription RNA nucleotides are added to the 3' end of the growing RNA stand.

A mutation in a scaffold protein that prevents interaction with the MAP kinase module is likely to have the following effect:

1. Unregulated activation of multiple signaling pathways 2. The signal would not be relayed to the correct effector protein

1. NLS 2. NES 3. helix with positively charged amino acids on one side and hydrophobic amino acids on the other side 4. N terminal hydrophobic signal sequence 5. mannose-6-phosphate 6. KDEL

1. nucleus 2. cytoplasm 3. mitochondria 4. ER 5. lysosome 6. ER

What effect would a loss of function mutation within the SH2 domain of a STAT protein have on the JAK-STAT signaling pathway? 1. STAT proteins would irreversibly dimerize, thus increasing gene expression 2. STAT proteins would remain in the cytoplasm preventing gene expression 3. STAT proteins would not be able to dimerize with one another, thus gene expression would be off

2 and 3

The following type of protein acts to turn on/off signaling by removing phosphate groups from tyrosines:

phosphatase

You are working on a viral protein called "A". In the presence of A, cellular protein "B" is found in the nucleus and cannot travel back out to the cytosol. Use your understanding of nuclear transport to select the most logical hypothesis for what protein A might be doing.

A is binding to B in the nucleus and obscuring the nuclear export signal on B

You are analyzing cells in the lab, and notice that a significant amount of Protein Disulfide Isomerases (PDI) are being secreted out of the cell through constitutive secretion. What is the most likely explanation for this occurrence?

A mutation in the DNA sequence has caused PDI to be translated without a proper KDEL sequence

Proteins are required for structure, function, and regulation within cells, organs, and tissues. Which of the following proteins is an example of a protein that is most directly involved in cell structure?

Actin

Which of the following proteins are most likely to be translated by a free ribosome found in the cytosol?

Actin

Signaling through a GPCR often results in an increase in the levels of small intracellular molecules or second messengers. The overall purpose of this is to:

Activate signaling molecules such as kinases

Select all statements that are correct regarding Wathelet et al:

Activation of antiviral signaling should result in phosphorylation of c Jun and IkB in control cells Activation of antiviral signaling resulted in phosphorylation of c Jun and IkB in Sendai Virus infected cells In this paper, Sendai virus is an activator of the antiviral response The main point of this paper is to demonstrate that SARS-CoV nsp1 inhibits the antiviral response

GPCRs signal through the activation of an associated G protein. This is similar to the regulation of importins and exportins by Ran. How?

All are inactivated by a GAP or GAP activity

You are working at a pharmaceutical company trying to develop a cancer drug that will inhibit the unfolded protein response (UPR). You reason that this drug can be given in combination with a drug that causes ER stress (causing massive protein misfolding) and may work synergistically to kill the cancer cell. Which of the following drugs would best inhibit the part or all of the UPR? (Focus on inhibition of the UPR, don't worry about possible toxicity, these things can be dealt with later)

An inhibitor of nuclear import A kinase inhibitor A protease inhibitor

Proteins have different parts, or domains, that have different functions. What is the function of an SH2 domain?

Binds to a phosphorylated tyrosine on a different protein

Proteins travel from the ER to the Golgi in COP-II coated vesicles. How is cargo incorporated into these vesicles?

Cargo is bound by a receptor that directly associates with COP II through an adaptor protein

Select the correct destination(s) for a receptor that enters the cell through the endocytic pathway (select all possible answers).

plasma membrane recycling endosome lysosome

1) _____________ ensure(s) correct targeting of vesicles and facilitates(s) the pairing of ________ to promote membrane fusion.

Rab proteins, SNAREs

How is nuclear cargo released, once inside the nucleus?

Ran-GTP binds importin to release cargo

Therapeutic proteins such as human insulin are produced on a large scale in E. coli, however the proteins produced by the bacteria are not functional until disulfides bonds are chemically added. Why is this?

E. coli do not have an ER

A protein with the amino acids K-D-E-L in the C-terminus of their protein sequence will be found in the following location most of the time:

ER

If arrestin is mutated in such a way as to inhibit interaction with a GPCR, the most likely outcome will be:

Endocytosis will be inhibited but the G protein will still be shut off

The following class of receptors either associates with a kinase or has a kinase domain:

Enzyme coupled receptors

The JAK-STAT pathway is a major activator of the antiviral response. In the presence of viral infection secretion of interferon by infected cells activates the Jak-STAT pathway. Dimerization of phosphorylated STAT results in:

Exposure of an NLS, import of the dimer into the nucleus and activation of gene expression

True or false: Proteins are generally imported co-translationally into the mitochondria.

False

Which of the following statements are true regarding the concept of gene expression?

Gene of interest is bound at the promoter by transcription factors and RNA polymerase, copied to RNA, then translated to protein.

If there is a mutation in importin that prevents it from interacting with the NLS of cargo proteins, what affect, if any, would this have on the UPR (unfolded protein response).

Gene regulatory protein would not be imported into the nucleus thus preventing transcription of UPR response genes

In class we spoke about the secretory pathway and how proteins enter the secretory pathway via import into the ER. After proteins are translated and correctly folded in the ER, where do they go? Select the immediate next step.

Golgi

In the lecture, I spoke about the secretory pathway and how proteins enter the secretory pathway via import into the ER. After proteins are translated and correctly folded in the ER, where do they go? Select the immediate next step.

Golgi

Removal of the inhibitor of NFkB, IkB, allows the gene regulatory protein to enter the nucleus and stimulate transcription. Select the correct mechanism of inhibition of NFkB by IkB?

IkB masks an NLS on NFkB

Proteins are required for structure, function, and regulation within cells, organs, and tissues. Which of the following proteins is an example of a protein that is most directly involved in immune function?

Immunoglobulin G

You are studying SARS-CoV-2 entry into host cells. You hypothesize that this virus enters cells using a mechanism similar to that used by SARS-CoV-1. You set up an experiment similar to the one the authors conducted in Wang 2008. Using Vero cells and GFP - SARS-CoV-2 you evaluate virus infectivity under the following conditions:1. Vero cells alone2. Vero cells + SARS-CoV-2 3. Vero cells + SARS-CoV-2 + chloroquineYou get the following results. What is your interpretation of the results? Do they support your hypothesis?

Inhibition of endosome acidification with chloroquine treatment had no effect on the infectivity of SARS-CoV2. SARS-CoV2 must use a different mechanism for entry than SARS-CoV1. My hypothesis was incorrect and needs to be modified based on these data.

What can you infer about the structure and cellular localization of the protein below based on its hydropathy index?

It is a multi-pass transmembrane protein that will be ultimately found on the cell surface but may also be found In the ER, Golgi, or lysosomal membranes.

Sometimes the proteins that help proteins to fold in the ER accidently travel to the Golgi. How are these ER resident proteins returned to the ER?

KDEL and the KDEL receptor

Which of the following are an example of receptor mediated endocytosis?

LDL receptor

Ras is a GTP binding protein that acts to relay signals from a receptor to different pathways. It is always bound to the plasma membrane. How is it activated?

Ligand binding results in phosphorylation of the receptor and recruitment of a GEF

You are studying a protein that you observe to be located in the ER and Golgi. You hypothesize that this protein is an ER resident protein. How would you go about testing your hypothesis?

Look for and remove KDEL sequence and look to see if the protein is secreted from cell Increase the pH of the Golgi and see if the protein is secreted from the cell. Look for and remove KDEL sequence and look to see if the protein is present in the ER.

Which of the following is NOT a factor involved in the process of nuclear transport?

M6P

Your dog is diagnosed with I-cell disease. One diagnostic indicator of this disease is high acid hydrolase levels are detected in the blood and urine. This symptom of the disease is most likely due to a defect in:

Mannose - 6 - phosphate receptor function

You are studying pancreatic cancer. Since Ras is frequently mutated in cancer, you decide to sequence the Ras gene in cancer cells to check for different mutations. You find a common mutation in your tumor samples that results in a Gly to Val mutation at position 12. A quick look at the literature tells you that this mutation falls into a GTPase domain. Based on this information, which of the following is a logical effect of this mutation on Ras protein function?

Mutant Ras cannot hydrolyze GTP resulting in constitutive Ras activation

You are studying SARS-CoVb. You hypothesize that upon entry to the host cell, the virus escapes from the endosome by inhibiting endosome acidification. You are trying to determine which viral protein is responsible for inhibiting endosome acidification. You have recently read a paper where they use pH-sensitive GFP to measure pH in different organelles. You decide to use pHlourin attached to an endosomal protein to measure the pH in the endosome in the absence and presence of different viral proteins. Which protein is responsible for inhibiting endosome acidification?

N

If the pinsulin receptor, an example of an integral membrane protein, is cotranslationally insertedinto the ER membrane with the C-terminus on the cytosolic side of the membrane and the N-terminus in the lumen of the ER, which side of the protein would you expect to bind to the ligand, pinsulin, when the protein is found on the cell surface?

N-terminal end of the protein will bind pinsulin

Which of the following is/are an example of a latent gene regulatory protein that is regulated by cleavage or proteolysis?

NFKB

Nuclear import receptors interact with the following part of the protein to be imported to the nucleus:

NLS

You are studying SARS-CoV2. You identify a viral protein that results in the decrease of MHC class I, a cell surface receptor, on the cell surface. You are trying to determine the mechanism for how the virus is decreasing cell surface MHC I. You decide to do an experiment to determine whether the receptor is making it all of the way through the secretory pathway. You take two samples of cells. 1. Uninfected 2. Infected with SARS-CoV2 You treat them with EndoH and analyze MHC I on a western blot and get the following results: 1. Uninfected cells - 50% of protein is endo H sensitive and 50% is Endo H resistant 2. SARS-CoV2 infected cells - 100% of protein is Endo H sensitive How would you best interpret these results? Is MHC I going all the way through the secretory pathway in infected cells?

No, MHC I is not going all the way through the secretory pathway. 100% of the protein is EndoH sensitive in infectedcells, this means that the virus is somehow inhibiting the protein from making it through the Golgi.

You are working in a colon cancer research lab. You are trying to identify new mutations in patient tumor samples. You identify a mutation in LRP6 that prevents binding to GSK3. Will this mutation promote cancer? Why

No, this mutation will result in degradation of B catenin and transcription will be off

If phosphorylation of an intracellular signaling molecule promotes signaling, then which enzyme would you expect to turn off signaling?

Phosphatase

Kinases can be either activating or inhibitory. You are working with a receptor and notice that when it is phosphorylated at Tyr135, gene expression increases. However when it is phosphorylated at Ser12 gene expression appears to be turned off. Which of the following are logical explanations for your observations?

Phospho-tyr135 recruits an activator of signaling, phosphor-ser12 recruits an inhibitor of signaling

You are a first year maternal fetal medicine fellow. A patient's 20 week ultrasound comes back with many anomalies and you decide to send the mother for fetal genetic testing to rule out severe genetic abnormalities. The report comes back with a mutation in the IP3 gated Ca++ chanel in the IP3 binding domain. Which of the following will NOT be affected by this mutation?

Phospholipase C beta activation

PI3 Kinase can be activated by Ras, GPCRs and RTKs. Select the best function of PI3 kinase from the choices below:

Phosphorylates inositol phospholipids on the plasma membrane to create binding sites for other signaling molecules

You are studying the development of frog embryos and you observe a brief expression of a cAMP-responsive gene after initial receptor stimulation. However, after the 30s of receptor stimulation, there is no gene expression (even in the presence of ligand). Which of the following explains the observed decrease in gene expression?

Phosphorylation of the receptor's cytoplasmic tail and recruitment of arrestin

You are studying the development of frog embryos and you observe a brief expression of a Ca++-responsive gene after initial receptor stimulation. However, after the 30s of receptor stimulation, there is no gene expression (even in the presence of ligand). Which of the following explains the observed decrease in gene expression?

Phosphorylation of the receptor's cytoplasmic tail, recruitment of arrestin, and pumping Ca++ into the ER

The activity of a protein can be regulated by controlling its cellular localization. Gene regulatory proteins or transcription factors are, for the most part, regulated this way. Phosphorylation of a protein's nuclear localization signal by a kinase will most likely have the following outcome:

Protein will not bind to importin and will be excluded from the nucleus

You are studying the secretory pathway. Working in the lab, you engineer a mutation into the gene encoding syntaxin7 preventing interaction with v-SNARES. Predict the most likely direct cellular consequence of this mutation?

Proteins that work in the lysosome, ie. acid hydrolases, will not be delivered to the lysosome

Many different cellular processes are regulated by GTP binding proteins - a kind of molecular switch. Their activity is regulated by their GTP/GDP binding status. Select the correct function and activity of Rab-GDP.

Rab in its GDP bound state is inactive and unable to participate in vesicle transport

Many different cellular processes are regulated by GTP binding proteins - a kind of molecular switch. Their activity is regu;ated by their GTP/GDP binding state. Select the correct function and activity of Rab-GTP.

Rab in its GTP bound state is active and able to participate in vesicle transport

Anabolic steroids mimic the natural hydrophobic steroid hormone testosterone and activate gene expression that results in muscle and bone growth. Once anabolic steroids bind to their receptors the following sequence of events happen:

Receptor conformational change, release of inhibitors, recruitment of activators, exposure of DNA binding domain, entry of receptor to nucleus, activation of gene expression

You are studying a new receptor; you believe it is involved in regulating expression of genes associated with cytokine secretion in the presence of gram negative bacteria. You decide to construct a kinase domain mutant of the receptor to test your hypothesis. If your hypothesis is correct, you should see the following result with your mutant.

Receptor phosphorylation should be inhibited in the presence of the bacteria and cytokines should be absent from the cell culture media

Secretion of histamine in response to pollen is an example of:

Regulated secretion

In recitation, we read a paper by Frieman et al that described a mechanism for how SARS-CoV1 inhibits the antiviral response. Select the correct summary of their findings.

SARS CoV 1 inhibits the antiviral response through the activity of ORF6. ORF6 binds to importins and tethers them to the ER/Golgi membrane. This prevents them from importing STAT1 into the nucleus.

1) While studying the secretory pathway, you make the following observation about proteins destined for the ER: Proteins translated in vitro in the absence of microsomes migrate more slowly on an SDS PAGE gel than those translated in the presence of microsomes. What might account for the observed protein size difference?

Signal peptidase, present only in the microsomes, cleaves the signal sequence from the protein, making the protein smaller and therefore migrate more quickly.

Receptor downregulation often occurs via endocytosis as a means of turning off a signaling pathway. How does receptor-mediated endocytosis result in turning off signaling?

Signaling is shut off rapidly through the budding of endosomes into themselves forming multivesicular bodies. This sequesters the cytoplasmic tail from intracellular signaling proteins.

Import of proteins into the mitochondrial matrix requires the following factors: (select all that apply)

TOM complex HSP70 TIM complex ATP

Select the best explanation of the unfolded protein response (UPR):

The UPR senses ER stress in the form of high levels of misfolded proteins and signals the cell to halt translation and induces expression of ER chaperones

Termicin is a small antifungal protein in termites that is produced by cells and secreted into termite saliva in response to a pathogen. In vitro translation of the termicin-encoding gene is performed, and the effects of that product are compared to those of termicin extracted from a termite. You see that extracted termicin exhibits more antifungal behavior than in vitro translated termicin. After further analysis, you see that extracted termicin contains 3 disulfide bonds, while in vitro translated termicin contains zero. The addition of microsomes to the in vitro translation reaction results in termicin with all 3 disulfide bonds. What experimental condition is most likely responsible for this difference?

The absence of microsomes prevented disulfide bond formation and rearrangement due to the absence of protein disulfide isomerase

You are studying a new retrovirus. The viral protein (X) appears to play a role in the export of the viral genomes to the cytoplasm. Protein X brings viral DNA to the cytoplasm and returns back to the nucleus after genome export is complete. Researchers have developed a new drug for the virus. Following treatment with the new drug, the viral protein stays in the nucleus and cannot export the viral genomes. What is the most plausible and logical function of the drug? Use your knowledge of nuclear transport to answer this question.

The drug inhibits the binding of Ran-GTP to the nuclear export receptor in nucleus.

The following is/are possible outcome(s) arising from a defect in the function of calnexin is:

The exit of misfolded proteins from the ER, bypassing quality control Increased ERAD Activation of the unfolded protein response

Why don't enzyme coupled receptors dimerize in the absence of ligand?

The ligand binds to both receptors, inducing dimerization and activation.

The LDL receptor is internalized via endocytosis every ten minutes and recycled to the membrane. How does the cell recycle the receptor and not the ligand (in this case LDL)?

The low pH (~5) of the early endosome causes dissociation of LDL from the receptor and the receptor is segregated into recycling endosomes.

You are studying Notch signaling in epithelial cells. You identify what you believe in an NLS in the cytoplasmic tail of the Notch receptor. You decide to mutate the NLS and study the phenotype. Evaluate the results below. Select the one that would NOT be expected in the NLS mutant.

The mutant receptor will not be cleaved following interaction with Delta ligand

You are doing an experiment in the lab and get the following results: Mutation of the promoter of a specific gene results in a loss of gene expression. Which of the following statements best explains the reason for this result?

The mutation destroyed the transcription factor binding site, preventing RNA polymerase recruitment, resulting in no transcription.

You discover a mutation in Ran-GTP that prevents binding to the nuclear import receptor (importin) in the nucleus. How will this affect nuclear import/export function? (select the most immediate effect)

The nuclear import receptor will not release the cargo protein in the nucleus

You are taking BIOL410 with Dr. Hemm and he gives you a list of proteins with no known function and tells you to determine which proteins are integral membrane proteins. Which of the following will tell you whether a protein is an integral membrane protein?

You run a hydropathy plot an look for hydrophobic peaks that span 20-30 amino acids

Protein structure is directly related to function. Which of the following statements is true regarding this concept ?

The primary structure consists of the amino acid sequence. The secondary and tertiary structures are dependent on interactions between the amino acid side chains. Mutations resulting in changes one or more amino acids has the potential to destroy the structure, and therefore the function, of the protein.

Select the correct outcome for a protein with a transmembrane domain. SRP binds to the SRP receptor, then:

The protein will be co-translationally embedded in the ER membrane

Different proteins have different functions and must be localized to the appropriate location in the cell. We discussed the function of NF-AT in the lecture and in recitation. Based on the function of this gene and the info provided in the entry, select the following logical assumptions that can be made. Select all statements that could be correct.

The sequence for NF-AT contains a short stretch of Lysine and Arginines NF-AT has an NLS NF-AT interacts with a nuclear import receptor

You are working in the lab studying BiP, an ER chaperone. You observe an accumulation of BiP in the Golgi. You suspect that there is something preventing it from returning to the ER. You decide to look at calnexin and calreticulin localization as controls and detect them mostly in the ER. Which of the following can explain why BiP is not returning to ER?

There is a mutation in the KDEL sequence in BIP

You observe that Ras is not activated even though the ligand is bound to the receptor. What could be the most likely mutation(s) responsible for this?

There is a mutation in the adaptor protein so it cannot recruit Ras-GEF There is a mutation in the SH2 domain of the adaptor protein so it cannot bind to the phosphorylated receptor There is a mutation in the kinase domain of the receptor.

You are working in the lab studying the translation of SARS-CoV-2 spike protein in hopes of making a mutant virus that is unable to enter cells through binding to the ACE2 receptor. You observe that a mutation has arisen within the viral genome that results in a change in the AUG codon in the Spike mRNA. The sequence is changed from AUG to AUA . What affect do you expect this mutation to have on Spike gene expression?

There will be Spike mRNA production but no protein translation due to the loss of the start codon. This virus will not be infectious.

Which of the following is NOT TRUE about nuclear receptors?

They are located on the surface of the nuclear envelope

You are studying mouse coronavirus nsp6 with protein sequence:>sp|P0C6X9|3636-3921SKRTRVIKGTCCWILASTFLFCSIISAFVKWTMFMYVTTHMLGVTLCALCFVSFAMLLIKHKHLYLTMYIMPVLCTFYTNYLVVYKQSFRGLAYAWLSHFVPAVDYTYMDEVLYGVVLLVAMVFVTMRSINHDVFSIMFLVGRLVSLVSMWYFGANLEEEVLLFLTSLFGTYTWTTMLSLATAKVIAKWLAVNVLYFTDVPQIKLVLLSYLCIGYVCCCYWGILSLLNSIFRMPLGVYNYKISVQELRYMNANGLRPPRNSFEALMLNFKLLGIGGVPVIEVSQIQYou use the Protscale and TMHMM to analyze the protein sequence to get an idea about the function of the protein.You get the following hydropathy plot and transmembrane analysis. Select all of the correct assumptions you can make based on this data. Select all assumptions supported by this data and your knowledge of the process of protein import to the ER.

This protein is an integral membrane protein This protein most likely binds SRP This protein most likely has a signal sequence This protein most likely has 6 or 7 transmembrane domains

PI-3 Kinase phosphorylates phospholipids. What is the purpose of this kinase activity?

To create binding sites for the recruitment of signaling proteins to the plasma membrane

The process of copying a DNA sequence to RNA is called:

Transcription

The process of synthesizing a polypeptide chain from an mRNA transcript is called:

Translation

True or False: Mitochondrial proteins have a signal sequence that is cleaved after import into the mitochondria.

True

You are studying a novel viral protein. There is nothing known about the function of this protein. You analyze the protein sequence you observe an N-terminal sequence that looks like a signal sequence. To determine whether this sequence is in fact a signal sequence you clone the gene and make a mutation in the sequence, changing all of the amino acids in that region to alanine. You then look to see if the protein binds SRP and is brought to the ER. If your hypothesis is correct and it is in fact a signal sequence you should get the following results to your experiment. Select the outcome that supports your hypothesis. WT = wild-type, unmutated protein M = mutant, protein with signal sequence mutated to alanines

WT protein binds to SRP, localizes to ER M protein does not bind to SRP, is found in the cytosol

Kinases and phosphatases turn proteins on and off through the following mechanism:

adding and removing phosphate groups, resulting in changes in protein conformation or interaction with other proteins

Which of the following amino acids would you expect to find in a transmembrane domain?

alanine, valine, isoleucine, leucine

Which of the following proteins are most likely to be translated by a ribosome found attached to the endoplasmic reticulum?

growth factor

Proteins have different parts, or domains, that have different functions. What is the function of a kinase domain?

has the ability to phosphorylate

Which of the following are NOT TRUE about G-protein coupled receptors (GPCRs)?

have kinase domains that get activated following ligand binding

Proteins are required for structure, function, and regulation within cells, organs, and tissues. Which of the following proteins is an example of a protein that is most directly involved in amino acid metabolism?

phenylalanine hydroxylase

Select the correct explanation of how integral membrane proteins arrive at the plasma memberane:

integral membrane proteins have a signal sequence and/or stop transfer sequences that are recognized by SRP. SRP brings the polypeptide + ribosome to the ER where translation continues into the ER until a stop transfer sequence or long stretch of hydrophobic amino acids is recognized and translation stops as the transmembrane domain of the protein moves the ER membrane. The ultimate destination of the ER membrane is the plasma membrane

Different proteins function in different places in the cell. Proteins that enter the secretory pathway are destined for specific locations in the cell. Select the CORRECT destination(s) for proteins that enter the secretory pathway.

lysosome, secreted from the cell, ER, plasma membrane

Choose the statement that best describes how proteins move between different compartments within the secretory and endocytic pathways.

proteins are packaged in vesicles that bud out of one membrane and fuse with the membrane at the next destination

ERAD, or ER associated degradation, is a quality control measure with the following function:

proteins that cannot fold correctly are ejected from the ER through the translocon and degraded by the ubiquitin-proteasome system in the cytosol

What is the fate of an acid hydrolase that does not get tagged with mannose-6-phosphate in the Golgi?

secreted out of the cell

Choose the correct function of tRNA.

tRNAs bring amino acids to the ribosome for addition to the growing polypeptide chain

Select the correct outcome for a protein lacking a transmembrane domain. SRP binds to the SRP receptor, then:

the protein will be translated into the lumen of the ER

The purpose of the signal sequence is:

through binding to the SRP, the signal sequence directs proteins destined for the secretory pathway to the ER