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Questions and Answers
What is the primary role of microsomes in mRNA translation?
What is the primary role of microsomes in mRNA translation?
What does the observation of a larger protein produced in the absence of microsomes indicate?
What does the observation of a larger protein produced in the absence of microsomes indicate?
Which component is responsible for recognizing and binding to the signal sequence during translation?
Which component is responsible for recognizing and binding to the signal sequence during translation?
What is the function of GTP hydrolysis in the cotranslational import process?
What is the function of GTP hydrolysis in the cotranslational import process?
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What role does the signal sequence play in the transport of proteins into the ER?
What role does the signal sequence play in the transport of proteins into the ER?
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What happens to the signal sequence during the translation process once it reaches the translocator?
What happens to the signal sequence during the translation process once it reaches the translocator?
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How would a mutation in the signal sequence affect protein targeting to the ER?
How would a mutation in the signal sequence affect protein targeting to the ER?
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Which complex is primarily involved in threading the growing polypeptide chain into the ER lumen?
Which complex is primarily involved in threading the growing polypeptide chain into the ER lumen?
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What is the role of GEF in the vesicle formation process?
What is the role of GEF in the vesicle formation process?
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What triggers the disassembly of the COPII coat from the vesicle?
What triggers the disassembly of the COPII coat from the vesicle?
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How do v-SNARE and t-SNARE proteins contribute to vesicle targeting?
How do v-SNARE and t-SNARE proteins contribute to vesicle targeting?
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What is the role of NSF in vesicle-membrane fusion?
What is the role of NSF in vesicle-membrane fusion?
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What is the function of the KDEL sequence in ER-resident proteins?
What is the function of the KDEL sequence in ER-resident proteins?
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How does the KDEL receptor facilitate the retrieval of ER-resident proteins?
How does the KDEL receptor facilitate the retrieval of ER-resident proteins?
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What happens when there are mutations in the LDL receptor gene?
What happens when there are mutations in the LDL receptor gene?
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What role does clathrin play in receptor-mediated endocytosis?
What role does clathrin play in receptor-mediated endocytosis?
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What is the primary function of Ras protein in the MAP kinase pathway?
What is the primary function of Ras protein in the MAP kinase pathway?
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Which subunits make up heterotrimeric G proteins?
Which subunits make up heterotrimeric G proteins?
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What effect does increased Ras activity have on cell proliferation?
What effect does increased Ras activity have on cell proliferation?
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What activity is primarily initiated by adenylyl cyclase in signal transduction?
What activity is primarily initiated by adenylyl cyclase in signal transduction?
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What two second messengers are produced from the action of phospholipase C (PLC)?
What two second messengers are produced from the action of phospholipase C (PLC)?
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How does β-arrestin downregulation affect GPCR phosphorylation?
How does β-arrestin downregulation affect GPCR phosphorylation?
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What is the role of PKC in cell signaling?
What is the role of PKC in cell signaling?
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What happens to receptor internalization when β-arrestin levels are low?
What happens to receptor internalization when β-arrestin levels are low?
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What role does signal peptidase play in protein processing within the ER?
What role does signal peptidase play in protein processing within the ER?
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Which mutation would most likely lead to mislocalization of a protein from the ER?
Which mutation would most likely lead to mislocalization of a protein from the ER?
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What is the purpose of creating fusion proteins with a reporter gene in determining ER localization signals?
What is the purpose of creating fusion proteins with a reporter gene in determining ER localization signals?
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How can the hydrophobicity profile of a protein sequence predict its integration into the ER membrane?
How can the hydrophobicity profile of a protein sequence predict its integration into the ER membrane?
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What triggers the Unfolded Protein Response (UPR) in cells?
What triggers the Unfolded Protein Response (UPR) in cells?
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Which component of the UPR is primarily responsible for initiating the translation of stress response genes?
Which component of the UPR is primarily responsible for initiating the translation of stress response genes?
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What is the function of Ran-GTP in cargo release within the nucleus?
What is the function of Ran-GTP in cargo release within the nucleus?
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What triggers the dissociation of the importin α/β-Ran-GTP complex in the cytoplasm?
What triggers the dissociation of the importin α/β-Ran-GTP complex in the cytoplasm?
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What is one consequence of having a defective signal peptidase?
What is one consequence of having a defective signal peptidase?
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What is the role of exportin in the nuclear export mechanism?
What is the role of exportin in the nuclear export mechanism?
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Which downstream action can occur as a result of IRE1 activation in the UPR?
Which downstream action can occur as a result of IRE1 activation in the UPR?
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Which complex is responsible for translocating proteins across the inner mitochondrial membrane?
Which complex is responsible for translocating proteins across the inner mitochondrial membrane?
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What is the consequence of GTP hydrolysis in the context of nuclear export?
What is the consequence of GTP hydrolysis in the context of nuclear export?
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How does the importin α/β-Ran-GTP complex return to the cytoplasm?
How does the importin α/β-Ran-GTP complex return to the cytoplasm?
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What happens to mitochondrial matrix proteins after entering the matrix?
What happens to mitochondrial matrix proteins after entering the matrix?
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Which part of the nuclear export mechanism involves the formation of a trimeric complex?
Which part of the nuclear export mechanism involves the formation of a trimeric complex?
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Study Notes
mRNA Translation and Protein Targeting to the ER
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ER Role: The endoplasmic reticulum (ER) plays a crucial role in protein processing and targeting.
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Signal Sequence: A signal sequence, a stretch of hydrophobic amino acids at the N-terminus of a newly synthesized polypeptide, directs proteins to the ER.
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Microsomes: Microsomes (rough ER vesicles) are essential for signal sequence removal and protein processing. They contain the machinery required for these steps.
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Signal Hypothesis: The Signal Hypothesis proposes that a specific amino acid sequence, known as the signal sequence, directs proteins to the ER.
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Signal Sequence Function: The signal sequence is recognized by the Signal Recognition Particle (SRP), pausing translation and preventing premature folding.
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SRP and SR: The SRP, in turn, binds to the SRP receptor (SR) on the ER membrane, guiding the ribosome to the translocator.
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Translocator/Sec61 Complex: The translocator, also known as the Sec61 complex, is a protein channel embedded in the ER membrane through which the growing polypeptide chain is threaded into the ER lumen.
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Signal Peptidase: Within the ER lumen, a protein called signal peptidase cleaves the signal sequence from the polypeptide chain, releasing the mature protein into the ER lumen.
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Mutations and their effects: Mutations in the signal sequence, SRP, SR, translocator, or signal peptidase can disrupt protein targeting and processing.
Experiment to Determine ER Localization Signal
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Fusion Proteins: Create fusion proteins by attaching different segments of the protein of interest to a reporter gene, like GFP (green fluorescent protein).
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Expression: Introduce these fusion protein constructs into cells.
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Observation: Observe the localization of the reporter protein using fluorescence microscopy. If the reporter protein is localized to the ER, the segment of the protein fused to it contains the ER localization signal.
Determining ER Membrane Insertion
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Stop-Transfer Sequences: Proteins destined for insertion into the ER membrane typically contain hydrophobic stretches of amino acids called stop-transfer sequences, in addition to their signal sequence.
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Predicting Insertion: By analyzing the hydrophobicity profile of a protein sequence, one can potentially predict the presence and location of transmembrane domains, indicating whether and how the protein will insert into the ER membrane.
Unfolded Protein Response (UPR)
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UPR Trigger: The UPR is triggered by the accumulation of unfolded or misfolded proteins in the ER lumen.
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IRE1, PERK & ATF6: IRE1, PERK, and ATF6 are three key proteins involved in triggering the UPR.
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IRE1: IRE1, a transmembrane kinase, activates downstream signaling pathways to reduce the burden of unfolded proteins.
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PERK: PERK, another transmembrane kinase, phosphorylates eIF2α, inhibiting protein synthesis and reducing the load of unfolded proteins.
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ATF6: ATF6, a transcription factor, translocates to the Golgi where it is cleaved and released to the nucleus to activate genes involved in the UPR.
Vesicular Transport and GEF-GAP Regulation
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GEF and Sar1: GEF (Sec12) activates Sar1 by exchanging GDP for GTP, enabling Sar1 to anchor to the ER membrane and recruit COPII coat proteins that form transport vesicles.
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GAP (Sec23): GAP (Sec23) stimulates GTP hydrolysis on Sar1, returning it to an inactive form, which causes COPII coat disassembly and prepares the vesicle for fusion with the Golgi.
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Importance of GEF-GAP Regulation: This precise GEF-GAP regulation is crucial for efficient vesicle formation and transport.
SNARE Complexes and Vesicle Fusion
- v-SNAREs and t-SNAREs: v-SNARE (vesicle-SNARE) proteins on transport vesicles recognize and bind to complementary t-SNARE (target-SNARE) proteins on the target membrane. This interaction ensures that the vesicle fuses with the correct target membrane.
- NSF: NSF (N-ethylmaleimide-sensitive factor) is an ATPase that plays a crucial role in disassembling the SNARE complex after membrane fusion. This disassembly is essential for recycling the SNARE proteins for subsequent vesicle fusion.
KDEL Sequence and ER Protein Retention
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KDEL Sequence: The KDEL sequence (Lys-Asp-Glu-Leu) is found at the C-terminal end of proteins that reside in the ER.
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KDEL Receptor: The KDEL receptor, located in the Golgi apparatus, recognizes and binds to the KDEL sequence of ER-resident proteins.
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Retrieval Mechanism: This binding triggers the packaging of the receptor-protein complex into COPI-coated vesicles, which return to the ER. This mechanism ensures that proteins essential for ER function are retrieved and maintained within the ER lumen.
LDL Receptor-Mediated Endocytosis
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LDL (low-density lipoprotein) particles: LDL particles, carrying cholesterol, bind to specific LDL receptors on the cell surface.
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Coated Pits: These receptors cluster in coated pits, which invaginate and pinch off to form clathrin-coated vesicles containing the LDL particles.
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Lysosome Fusion: The vesicles fuse with lysosomes, where the LDL particles are degraded, releasing cholesterol for cellular use.
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Defects in Receptor-Mediated Endocytosis: Defects in any of the steps in receptor-mediated endocytosis can lead to impaired LDL uptake, potentially causing conditions like familial hypercholesterolemia (high cholesterol levels).
Nuclear Import and Export Mechanisms
Nuclear Import
- Nuclear Localization Signal (NLS): Proteins destined for the nucleus contain a Nuclear Localization Signal (NLS).
-Importin α and β: Importin α and β, proteins residing in the cytoplasm, recognize and bind to the NLS.
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Nuclear Pore Complex (NPC): The importin α/β-cargo protein complex interacts with FG repeats of nucleoporins and moves through the NPC.
- Ran-GTP: Inside the nucleus, Ran-GTP binds to importin β, releasing the cargo protein into the nucleus.
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Recycling: The importin α/β-Ran-GTP complex is exported back to the cytoplasm, where Ran-GAP stimulates GTP hydrolysis on Ran, causing dissociation of the complex and allowing the importins to be used again.
Nuclear Export
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Nuclear Export Signal (NES): Proteins destined for export from the nucleus contain a Nuclear Export Signal (NES)
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Exportin (Crm1): Exportin, such as Crm1, binds to the NES on the cargo protein and forms a complex with Ran-GTP
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NPC Translocation: The export complex interacts with FG repeats of nucleoporins and moves through the NPC.
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Cargo Release in the Cytoplasm: Upon reaching the cytoplasm, Ran-GAP stimulates GTP hydrolysis on Ran, resulting in dissociation of the export complex and release of the cargo protein.
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Recycling: Exportin and Ran-GDP are recycled back to the nucleus for further export.
Mitochondrial Protein Import and Transport
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TOM Complex: Proteins destined for the mitochondrial matrix initially pass through the TOM complex located on the outer mitochondrial membrane.
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TIM23 Complex: The protein then moves through the TIM23 complex located on the inner mitochondrial membrane.
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Mitochondrial Processing Peptidase (MPP): Once inside the matrix, the targeting signal is cleaved by MPP.
Heterotrimeric G Proteins, PLC, & Adenylyl Cyclase Pathways
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Heterotrimeric G Proteins Role: Heterotrimeric G proteins act as molecular switches, transducing signals from activated GPCRs to downstream eNectors like adenylyl cyclase and PLC.
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G Protein Subunits: G proteins consist of α, β, and γ subunits.
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GDP/GTP Exchange: Upon GPCR activation, the α subunit exchanges GDP for GTP, activating downstream eNectors, and dissociates from the βγ subunits.
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Adenylyl Cyclase Pathway: Adenylyl cyclase catalyzes the conversion of ATP to cAMP, a second messenger that activates protein kinase A (PKA).
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PLC Pathway: PLC cleaves PIP2 (phosphatidylinositol 4,5-bisphosphate) into DAG (diacylglycerol) and IP3 (inositol 1,4,5-trisphosphate), both second messengers. DAG activates protein kinase C (PKC), while IP3 triggers calcium release from the ER.
β-Arrestin Downregulation of GPCR
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β-Arrestin Function: β-arrestins bind to phosphorylated GPCRs and can either desensitize the receptor or promote its internalization via clathrin-coated pits.
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Downregulation of β-arrestin: Downregulation of β-arrestin would potentially lead to reduced receptor phosphorylation and decreased receptor internalization.
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Potential Consequences: These changes could alter the signaling pathway and its downstream effects.
Ras Protein and MAP Kinase Pathway
- Ras Activity Effects: The Ras protein, a small GTP-binding protein in the MAP kinase pathway, activates MAPKKK when bound to GTP. Increased Ras activity leads to increased activation of MAPKKK, amplifying the signal and promoting cell proliferation. Conversely, decreasing Ras activity weakens the signal and inhibits proliferation.
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