Cell Bio

B

Certain cysteine-containing proteins of the mitochondrial intermembrane space are imported from the cytosol with the help of the Mia40 protein via a disulfide relay system. What drives the unidirectional import of these proteins? Are these proteins reduced or oxidized at their cysteine residues upon import? A. ATP hydrolysis; oxidized B. Theelectron-transportchain;oxidized C. ATP hydrolysis; neither oxidized nor reduced D. ATP hydrolysis; reduced E. The electron-transport chain; reduced

A. Proteins containing an NLS would be actively exported from the nucleus, while NES- containing proteins would be actively imported.

According to the model for nuclear transport described in this chapter, what do you think would happen if you could artificially limit all Ran-GAP activity to the nucleus and all Ran-GEF activity to the cytosol? A. Proteins containing an NLS would be actively exported from the nucleus, while NES- containing proteins would be actively imported. B. Both import and export of nuclear proteins would be stalled, as they lose their directionality. C. Protein import into the nucleus would be reversed, but export would be unaffected. D. Protein import into the nucleus would be stalled, but export would be unaffected. E. Nothing would change; this is the normal Ran-GAP and Ran-GEF distribution.

B. involved in the transport of proteins with the first signal sequence but not the second one.

A geneticist has devised a strategy to study protein translocation into the endoplasmic reticulum (ER) in yeast cells. She is interested in two different signal sequences that are thought to operate via slightly different translocation mechanisms. Using genetic engineering, she has fused the first signal sequence to a protein whose cytosolic expression is absolutely necessary for cell survival in the selective medium, but is inactive when in the ER. In the same cell, she has also fused the second signal sequence to a toxic protein whose cytosolic expression leads to cell lysis but is harmless when in the ER. Whereas wild-type cells undergo lysis upon the expression of these fusion proteins, she has been able to identify viable mutants, each of which has a loss- of-function mutation in a gene encoding a protein involved in membrane translocation. The products of these genes are probably ... A. involved in the general transport of proteins into the ER, regardless of the type of signal sequence. B. involved in the transport of proteins with the first signal sequence but not the second one. C. involved in the transport of proteins with the second signal sequence but not the first one. D. involved in the transport of proteins with a novel signal sequence (i.e. neither the first signal sequence nor the second one).

C. The protein is likely to be an ER resident, helping with the folding of nascent imported proteins.

A protein is covalently attached to glycosylphosphatidylinositol. Which of the following is typically NOT true regarding this protein? A. The linkage of the anchor to the C-terminus of the protein occurs in the ER. B. The attachment of the anchor coincides with cleavage of a C-terminal transmembrane segment of the protein precursor. C. The protein is likely to be an ER resident, helping with the folding of nascent imported proteins. D. A phospholipase can cleave the protein from the membrane. E. The anchor affects the localization of the protein in the membrane.

C. It has a much higher calcium ion concentration.

Compared to the cytosol, which of the following is generally true about the lumen of the endoplasmic reticulum in our cells? A. It has a higher pH. B. It has a more reducing environment. C. It has a much higher calcium ion concentration. D. It has a higher density of ribosomes. E. All of the above.

A. Cytosol

Consider a human cell such as a hepatocyte. Which of the following compartments occupies a larger volume in the cell? A. Cytosol B. Nucleus C. Endoplasmicreticulum D. Mitochondria E. Peroxisomes

D. Rough ER membrane

Consider a human liver hepatocyte. Among the following membranes, which one has the largest total area? A. Plasma membrane B. Nuclear inner membrane C. Mitochondrial outer membrane D. Rough ER membrane E. Smooth ER membrane

B. the transcription regulatory protein accumulates in the cytosol.

Consider a transcription regulatory protein that has both a nuclear localization and a nuclear export signal and is normally found both in the nucleus and in the cytosol at comparable concentrations. This protein has a high-affinity binding partner in the nucleus. Upon activation of a certain signaling pathway, the binding protein is ubiquitylated and degraded. As a result of this, ... A. the transcription regulatory protein accumulates in the nucleus. B. the transcription regulatory protein accumulates in the cytosol. C. the distribution of the transcription regulatory protein does not change, but the expression of its target genes may be altered. D. the distribution of the transcription regulatory protein does not change, and the expression of its target genes is not necessarily changed as a result of the degradation event.

D.

Consider a transmembrane protein with the following topology that has an internal signal sequence (helix 1). If you fuse a canonical ER signal sequence at the N-terminus of this protein, how would you expect the topology to change? The ER lumen is at the bottom in all drawings. For simplicity, assume that the effect of charged residues flanking the transmembrane helices is negligible in this case. N C Cytosol 12 ER lumen N C 12 A. C 12 N B. N C 12 C. 12 N C D. N 12 C E.

B

Consider two cells, A and B. Both are approximately spherical, but cell A is a bacterium with a diameter of only about 1 μm, while the diameter of the eukaryotic cell B is about 10 μm. If the plasma membrane in the eukaryotic cell comprises only about 2% of the total cell membrane, which cell has a higher ratio of total cell membrane to volume? Write down A or B as your answer.

A. I;2

Considering the following hydropathy plot for a multipass transmembrane protein in the plasma membrane, are the protein termini expected to be located inside (I) or outside (O) of the cell? Which of the regions indicated as 2 and 3 in the plot is expected to have more positively charged residues? The position of positively charged residues near the first transmembrane helix is indicated by an asterisk on the plot. +25 Hydropathy index -25 * 23 A. I;2 B. I;3 C. O;2 D. O;3

C. Phosphorylation within the nuclear export signal interferes with the function of the signal.

Cyclin B1, a key cell cycle regulatory protein in vertebrates, is mostly cytosolic before mitosis. Early in mitosis, however, the protein is phosphorylated by certain protein kinases and consequently accumulates in the nucleus. How can phosphorylation bring about nuclear accumulation of this protein? A. Phosphorylation within the nuclear localization signal inhibits the function of the signal. B. Phosphorylation within the nuclear export signal enhances the function of the signal. C. Phosphorylation within the nuclear export signal interferes with the function of the signal. D. Phosphorylation elsewhere on the protein enhances binding to cytosolic proteins.

methionine

Fill in the blank in the following paragraph regarding the recognition of tail-anchored proteins by their targeting machinery. DO NOT use abbreviations. "When the hydrophobic transmembrane α helix at the C-terminus of an ER tail-anchored protein emerges from the ribosome, the tail- anchored-binding domain (TABD) of the Get3 ATPase binds to it. The job of TABD in Get3 is similar to that of the flexible hydrophobic domain in the SRP54 protein of the signal-recognition particle, in that it also has to recognize a degenerate set of hydrophobic α- helices. Therefore, it is not surprising that, just like the corresponding domain in SRP54, the binding site in Get3 is rich in ... residues."

Endoplasmic Reticulum

Fill in the blank in the following paragraph. DO NOT use abbreviations. "A significant fraction of total membrane area in a eukaryotic cell encloses the lumen of the ..., which forms an extended netlike labyrinth of tubules and sacs. Secretory proteins are normally synthesized by ribosomes bound to a special type of this compartment."

E. Endoplasmic reticulum

Imagine a protein that has been engineered to contain a nuclear localization signal, a nuclear export signal, a C-terminal peroxisomal targeting sequence, and a canonical endoplasmic reticulum (ER) signal sequence. With all of these signals, where would you expect to find the protein after its synthesis? A. Cytosol B. Nucleus C. Shuttling between the cytosol and the nucleus D. Peroxisomes E. Endoplasmic reticulum

BDCA

In the following graph, the magnitude of concentration difference across the nuclear pore complexes (NPCs) is plotted for four molecules (A to D) as a function of time, starting from an arbitrarily chosen initial concentration difference. Indicate which curve corresponds to each of the following molecules. Your answer would be a four-letter string composed of letters A to D only, e.g. ABCD. 2 1 0 0 10 Time (min) A B C D Concentration difference across the nuclear envelope (nM) ( ) A large protein that is being actively transported across the NPC ( ) A small water-soluble molecule ( ) A small protein composed of a few dozen residues ( ) A large protein that is NOT actively transported into or out of the nucleus

D. Left; 0.1 μm

In the following schematic diagram of a nuclear pore complex, on which side is the cytosol located? What is the approximate diameter of the pore, as indicated by the vertical bar? A. Right; 1 μm B. Right;0.1μm C. Left;1μm D. Left; 0.1 μm

TFTF

Indicate true (T) and false (F) statements below regarding N-linked glycosylation of proteins. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) N-linked glycosylation can be carried out co-translationally, possibly at multiple asparagine residues on the same protein molecule. ( ) N-linked glycosylation is a gradual process, with step-by-step addition and trimming events that commence with the addition of N-acetylglucosamine to an asparagine side chain. ( ) Most proteins synthesized in the rough ER are N-glycosylated, and some of them require this modification for their correct folding. ( ) Once a protein is properly folded in the ER, its attached oligosaccharides are quickly removed by an N-glycanase, although it may be glycosylated again later.

TTTF

Indicate true (T) and false (F) statements below regarding peroxisomal proteins. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) All peroxisomal proteins are encoded in the nucleus. ( ) Some peroxisomal proteins are synthesized by ribosomes attached to the rough endoplasmic reticulum. ( ) All peroxisomal proteins reach the organelle after their synthesis is completed. ( ) Peroxisomal proteins have to be unfolded before import.

FTFF

Indicate true (T) and false (F) statements below regarding the compartmentalization of cells. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) Almost all eukaryotic cells have plastids, but only plant cells have chloroplasts capable of photosynthesis. ( ) The perinuclear space is topologically equivalent to the extracellular space. ( ) The mitochondria and chloroplasts are thought to have evolved by invagination and pinching off from the plasma membrane of the ancient eukaryotic cell. ( ) All organelles in a eukaryotic cell can be constructed de novo, which means that the information to construct them is encoded in the genome.

FFFF

Indicate true (T) and false (F) statements below regarding the mitochondrial protein import system. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) Mitochondrial proteins should fold natively twice: once in the cytosol and once inside the organelle. ( ) β-barrel proteins that are abundant in the mitochondrial outer membrane are imported from the cytosol independently of the TOM complex. ( ) Signal sequences that target precursor proteins to the mitochondrial matrix form an α- helix in which positively charged residues cluster near its N-terminus, while uncharged or hydrophobic residues cluster near the other side. ( ) At least two signal sequences are required to direct proteins to the mitochondrial matrix.

TFTT

Indicate true (T) and false (F) statements below regarding the nuclear transport of proteins. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) Many nuclear import and export receptors are members of the same protein family. ( ) Most nuclear import receptors contain unstructured domains with FG-repeats. ( ) Adaptor proteins that simultaneously bind to nuclear localization signals and to importins are required for the import of some nuclear cargo proteins. ( ) Ran is mostly found in its GTP-bound form in the nucleus.

TTFT

Indicate true (T) and false (F) statements below regarding the nucleus and nuclear protein transport. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) The inner and outer nuclear membranes are continuous with each other, yet maintain distinct protein compositions. ( ) The outer nuclear membrane is studded with ribosomes engaged in protein synthesis. ( ) The endoplasmic reticulum lumen is continuous with the nuclear interior. ( ) Ribosomal proteins pass through the nuclear pore complexes twice; they are imported into the nucleus after synthesis, and are exported from the nucleus after assembly with ribosomal RNA.

IAAI

Indicate whether each of the following descriptions better matches the ATF6 (A), the IRE1 (I), or the PERK (P) branch of the ER unfolded protein response. Your answer would be a four-letter string composed of letters A, I, and P only, e.g. APII. ( ) It involves a noncanonical cytoplasmic splicing process. ( ) Its sensor is a latent transcription regulator. ( ) It involves regulated proteolysis of the sensor protein in the Golgi apparatus. ( ) Its sensor bears both kinase and endoribonuclease activities.

SSRSS

Indicate whether each of the following descriptions better matches the rough (R) or the smooth (S) endoplasmic reticulum (ER). Your answer would be a five-letter string composed of letters R and S only, e.g. SRSRR. ( ) It mostly has a tubular appearance. ( ) It contains the transitional ER. ( ) It is coated by ribosomes. ( ) It can be specialized for functions such as detoxification and lipid metabolism. ( ) Sarcoplasmic reticulum in muscle cells is one of its specialized forms.

CCMBB

Indicate whether each of the following descriptions refers to protein import into mitochondria (M), chloroplasts (C), or both (B). Your answer would be a five-letter string composed of letters M, C, and B only, e.g. BBBMC. ( ) ( ) ( ) ( ) ( ) ATP and GTP hydrolysis drive translocation into the organelle. The organelle has an extra compartment that requires extra signal sequences for protein targeting. Transport through the double membrane is driven in part by an H+ gradient across the inner membrane. Imported precursor proteins have amphiphilic N-terminal signal sequences that are usually removed after use. Hsp70 family chaperones inside the organelle assist in protein translocation during import.

CCGCC

Indicate whether each of the following occurs on the cytosolic side of the ER membrane (C), on the lumenal side of the ER membrane (L), or in the Golgi apparatus (G). Your answer would be a five-letter string composed of letters C, L, and G only, e.g. GGGLC. ( ) Assembly of GlcNAc oligosaccharides on dolichol phosphate ( ) Synthesis of phosphatidylcholine ( ) Synthesis of sphingomyelin ( ) Ubiquitylation of misfolded ER protein ( ) Synthesis of phosphatidic acid

GGTVT

Indicate whether each of the following transport processes occurs via the mechanisms described as gated transport (G), transmembrane transport (T), or vesicular transport (V). Your answer would be a five-letter string composed of letters G, T, and V only, e.g. VTTTG. ( ) Import into nucleus ( ) Export from nucleus ( ) Import into mitochondria ( ) Return from Golgi to ER ( ) Return from ER to cytosol

CNN

Indicate whether the C-terminus (C) or the N-terminus (N) of each of the following proteins is expected to be located in the cytosol upon membrane integration of the protein. Your answer would be a three-letter string composed of letters C and N only, e.g. CCC. ( ) A single-pass transmembrane protein that has one N-terminal signal sequence and one internal stop-transfer signal ( ) A single-pass transmembrane protein that has one internal signal sequence that is preceded by a patch of positively charged residues ( ) An ER tail-anchored protein

CCEEE

Indicate whether the location of each of the following is topologically equivalent to the cytosol (C) or the extracellular space (E). Your answer would be a five-letter string composed of letters C and E only, e.g. CCECE. ( ) Ribosomes ( ) Chromatin ( ) Lysosomal hydrolases ( ) Calcium ions in the ER ( ) Peroxisomal catalase

D. being expelled to the Golgi apparatus for disposal.

Misfolded proteins in the ER may actively undergo any of the following EXCEPT ... A. binding to chaperones such as BiP to allow unfolding and refolding. B. binding to chaperones such as calnexin to prevent aggregation. C. being transported back to the cytosol for degradation. D. being expelled to the Golgi apparatus for disposal. E. being glycosylated and binding to lectins.

D. BiP

Mitochondrial hsp70 is to matrix protein import what ... is to post-translational ER protein import. A. Ribosome B. Sec61 C. Sec63 D. BiP E. PDI

BAC

Proteins of the protein disulfide isomerase (PDI) family share common domains that harbor an active-site CXXC motif (C = cysteine; X = one of several residues). During the course of a redox reaction, one of the active-site cysteines in its reduced form can attack a disulfide bond in a substrate protein (that could itself be another PDI family member) to form a mixed disulfide between the enzyme and its substrate. This is then attacked by the other active-site cysteine, releasing the substrate in reduced form. The reverse of these reactions can occur instead in order to make disulfide bonds in target proteins. CXXA mutant PDI family proteins—in which one of the active-site cysteines is mutated to an alanine—can be trapped in the intermediate mixed disulfide state for an elongated time, facilitating the identification of their substrate proteins. Using these mutations in each of three interacting PDI family proteins—A, B, and C (one mutation at a time)—you have discovered that the mutant B interacts with A and C, while mutant A interacts with C but not with B. Finally, mutant C interacts with neither of the other two. The following diagram shows the thermodynamically favorable flow of electrons (plus protons) in the cascade involving these three proteins. Based on your results above, what proteins correspond to 1, 2, and 3 in the diagram, respectively? Your answer would be a three- letter string composed of letters A to C only, e.g. CBA.

C. It is normally assembled in the nucleus and exported to the cytoplasm by exportins.

Rough microsomes can be subjected to a "salt extraction" procedure in which a high salt concentration is used to remove membrane-associated ribosomes and peripheral proteins. Such salt-extracted microsomes are known to be translocation-incompetent, meaning that when present co-translationally in vitro, they fail to protect translated proteins from protease digestion. However, adding back an 11S particle (S is the sedimentation coefficient) purified from the salt- wash fraction is sufficient to restore the protein-translocation activity of the salt-extracted microsomes. Which of the following do you think is true regarding the 11S particle? A. It is composed of 21 proteins. B. It is a digestion product of ER-associated ribosomes. C. It is normally assembled in the nucleus and exported to the cytoplasm by exportins. D. It is an ER integral membrane protein that can interact with the translocon. E. It requires high salt concentration for its function in vivo.

BDAEC

Sort the following events as they occur during the peroxisomal protein import cycle, starting with the release of cargo from Pex5. Your answer would be a five-letter string composed of letters A to E only, e.g. ABEDC. (A)Pex5 deubiquitylation (B) Pex5 ubiquitylation (C) Docking and translocation of the cargo protein along with Pex5 (D)Pex5 export from the peroxisome with the help of ATPases Pex1 and Pex6 (E)Pex5 binding to a cargo protein containing a C-terminal peroxisomal targeting sequence

D. 2, 4, 6

The following diagram depicts the topology of a multipass transmembrane protein in the endoplasmic reticulum (ER) membrane. Which set of helices act as stop-transfer signals in this protein? N Cytosol C 1 2 3 4 5 6 ER lumen A. 1, 2, 3 B. 4, 5, 6 C. 1, 3, 5 D. 2, 4, 6 E. 1, 6

C. nuclear export, but not import.

The formation of a stable ternary complex involving Ran GTPase, a nuclear transport receptor, and a cargo protein occurs in ... A. both nuclear import and export. B. nuclear import , but not export. C. nuclear export, but not import. D. neither nuclear import nor export.

D. binds GTP.

The signal-recognition particle (SRP) ... A. is a heterodimeric protein. B. transientlyinhibitstranslationandpolypeptideelongationbybindingtoandinhibiting the elongation factors. C. accompanies the nascent polypeptide all the way into the ER lumen. D. binds GTP. E. is permanently attached to the cytosolic face of the ER membrane, thus bringing the ribosomes into close proximity of the translocon.

No

The signal-recognition particle is not the only factor that evaluates the authenticity of endoplasmic reticulum (ER) signal sequences in proteins. The Sec61 complex is also able to recognize the signal sequences and "opens" after binding to them. Even single point mutations within the signal sequence of a protein can render the protein unable to enter the ER efficiently, as the Sec61 complex does not readily open in response to the mutant sequence. However, "suppressor" mutations in genes encoding components of the translocation pathway, including the Sec61 subunits, can partially restore the wild-type localization of proteins with mutant signal sequences. Many such suppressor mutations (also called prl mutations) map to or near the "plug" domain in the Sec61 translocon. These mutations, including the deletion of the entire plug, generally result in destabilization of the closed conformation of the translocon and favor its open conformation. Your friend has mutated a certain residue in the plug domain of the yeast Sec61. She has just finished measuring the translocation efficiency of an ER protein with either a wild- type or a mutant (defective) signal sequence, either in wild-type or in her Sec61-mutant cells, and has obtained the following results. Based on these early results, does her Sec61 mutation show a prl phenotype? Write down Yes or No as your answer.

TTFF

Tom40 is a nuclear-encoded essential subunit of the TOM complex in the outer mitochondrial membrane. It is a β-barrel protein that forms the pore through which precursor proteins enter the intermembrane space from the cytosol. Indicate true (T) and false (F) statements below regarding the Tom40 protein. Your answer would be a four-letter string composed of letters T and F only, e.g. TTTF. ( ) Incorporation of new Tom40 in the outer membrane requires preexisting Tom40 in that membrane. ( ) Formation of new TOM complexes is dependent on the SAM complex. ( ) Tom40 is partially translocated through the outer membrane and is then transferred in the plane of the membrane to fold into its native conformation. ( ) Tom40 is translocated through the inner membrane as a precursor.

E. Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Ser- Ser-Arg-Tyr-Leu

Which of the following is NOT a likely ER signal sequence recognized by the signal- recognition particle? All sequences are written with their N-terminus on the left. A. Met-Lys-Leu-Ser-Leu-Val-Ala-Ala-Met-Leu-Leu-Leu-Leu-Ser-Ala-Ala-Arg-Ala B. Met-Glu-Met-Phe-Gln-Gly-Leu-Leu-Leu-Leu-Leu-Leu-Leu-Leu-Ser-Met-Gly-Gly- Thr-Trp-Ala C. Met-Lys-Ala-Lys-Leu-Leu-Val-Leu-Leu-Tyr-Ala-Phe-Val-Ala-Gly-Asn D. Met-Met-Ala-Ala-Gly-Pro-Arg-Thr-Ser-Leu-Leu-Leu-Ala-Phe-Ala-Leu-Leu-Cys- Leu-Pro-Trp-Thr-Gln-Val-Val E. Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Ser- Ser-Arg-Tyr-Leu

A

Which of the following proteins or protein complexes is directly required for the targeting of mitochondrial inner membrane multipass proteins, such as metabolite transporters, whose signal sequence is normally not cleaved after import? A. TIM22 B. TIM23 C. OXA D. Mia40 E. SAM

E. All of the above are possible scenarios and occur naturally.

Which of the following scenarios does NOT normally occur on a nuclear pore complex? A. A protein complex is imported into the nucleus, with ONLY one of its subunits containing a nuclear localization signal (NLS). B. In a single pore, an NLS-containing protein is imported, while at the same time a nuclear export signal (NES)-containing protein is exported. C. A nuclear import receptor is exported from the nucleus through the pore. D. A protein is imported on its own through the pore, without the need for a separate import receptor. E. All of the above are possible scenarios and occur naturally.

D

Which reaction is normally catalyzed by the enzyme catalase in the peroxisome? A. 2O2- + 2H+ → H2O2 + O2 B. FMNH2 + O2 → FMN + H2O2 C. C2H5OH (ethanol) + NAD+ → C2H4O (acetaldehyde) + NADH + H+ D. 2H2O2 → 2H2O + O2 E. Both C and D above

ABF

Which subset of the following is directly involved in driving protein import into the mitochondrial matrix space? Choose all correct sources. Your answer would be a string composed of letters A to G only, in alphabetical order, e.g. AE. A. ATP hydrolysis inside mitochondria B. A TP hydrolysis outside mitochondria C. GTPhydrolysisinsidemitochondria D. GTP hydrolysis outside mitochondria E. Light F. Membrane potential across the inner membrane G. Membrane potential across the outer membrane

A. X;1

You set up an in vitro translation system containing the entire translation machinery but devoid of any component of the endoplasmic reticulum (ER) targeting machinery. To this system, you can add mRNA encoding either a 20 kD secretory protein or a 20 kD cytosolic protein. You perform in vitro translation in the presence of radioactively labeled methionine, with or without the addition of saturating amounts of SRP or microsomes, as indicated below. After separating the protein products by SDS-PAGE, and visualizing the radioactivity by autoradiography, you obtain the following results. The presence or absence of each component in the reaction is indicated at the top of the corresponding lane(s) by + and -, respectively. The numbers on the left indicate the apparent molecular mass (×1000) of spots on the gel. Which protein (X or Y) is the secretory protein? Which of the reactions (1 or 2) contained SRP? 123 Microsomes - - + SRP ? ? + XmRNA + - + - + - YmRNA - + - + - + 20 - 17 - 8- A. X;1 B. X;2 C. Y;1 D. Y;2