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Where are the majority of mitochondrial proteins synthesized in the cell and how are they targeted specifically for incorporation into mitochondria?
Cytosol on cytoplasmic ribosomes. Mitochondrial- amino acid signal sequence at their N-terminus that a signal peptidase rapidly removes after import.
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What are the mechanisms called that halt the cell in various stages of the cell cycle to ensure that everything is working OK before continuing on? Hint: this is a ONE word answer.
Checkpoints.
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Cyclin dependent kinase is present at steady state levels throughout interphase and mitotic cyclin levels increase steadily throughout interphase. Why then, is the MPF not activated during G1 and S? Discuss ONE mechanism that keeps the already complexed cyclin/CDK complex from being activated until the cell is ready to enter mitosis. Be specific here please.
MPF is not activated because the phosphorylation/dephosphorylation of two sites activate it and deactivate. One site (T161) is phosphorylated by CAK which activates MPF. Another site (Y15), is phosphorylation by Wee1 which deactivates it. MPF can be reactivated by dephosphorylation by Cdc25 at the Y15 site.
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Name two second messengers that are involved in signaling pathways.
cAMP is a second messenger that is involved in the GPCR pathway. It made by upon activation of adenylnyl cyclase. cAMP activates PKA which head towards the nucleus.
- IP3 is involved in the inositol phospholipid signaling pathway. It is made upon cleavage of PI(4,5)P2 by the activated phospholipase.
- IP3 then opens IP3-gated Ca2+ on the ER which releases Ca2+.
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As a pharmacologist, you are studying a chemotherapy agent called colecytecase. Cells that are exposed to colecytecase enter mitosis normally and proceed to metaphase. However, the cells become arrested at this phase and fail to enter anaphase. Additionally, you note that cyclin levels remain elevated. What protein or proteins could colecytecase inhibit that would account for both of these observations?
- APC – which ubiquitinates cyclins.
- The APC/C catalyzes the ubiquitylation and destruction of two major typesof proteins. The first is securin, which protects the protein linkages that holdsister-chromatid pairs together in early mitosis. Destruction of securin in metaphaseactivates a protease that separates the sisters and unleashes anaphase, asdescribed later. The S- and M-cyclins are the second major targets of the APC/C.Destroying these cyclins inactivates most Cdks in the cell.
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Clathrin coated vesicles bud from eukaryotic plasma membrane fragments when adapter proteins, clathrin and dynamin GTP are added. What would you expect to observe if the following omissions were made to the experiment. Explain your answers. **It’s not enough to just say that pits would or would not form, you need to be detailed in your answer.
- Adaptor proteins were omitted
- No vesicles would form because adaptins link clathrin to the membrane
- Clathrin was omitted
- Adaptor proteins would still bind to receptors in the membrane but no clathrin coat would form. Thus no pits or vesicles would form.
- Dynamin was omitted
- Clathrin coated pits would form and proceed toward vesicle formation but membrane fusion is blocked with dynamin, so deep invaginated pits would form
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A secreted protein has begun translation on a cytoplasmic ribosome. The N-terminal sequence of the protein consists of 20 hydrophobic amino acids. During translation, the N-terminus is bound by the SRP. Describe the subsequent steps in the translation and targeting of the protein so that it becomes a soluble protein in the ER lumen.
When SRP binds to the N-terminus, translation is stalled.
SRP delivers ribsome/nascent protein complex to the SRP receptor on the ER membrane.
SRP docks onto SRP receptor which creates GTP binding domains.
GTP binds. Hydrolysis if 2 GTP molecules results in ribosome binding to and opening up the translocon particle.
- As polypeptide elongates, it moves into ER.
- Opening of translocon attracts signal peptidase and cleaves of the signal sequence
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What type of secretory vesicle takes proteins outside the cell?
Clatherin coeated vesicles
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The nuclear pores regulate traffic between the inside of the nucleus and the cytoplasm. List seven molecules that pass in and out of the nuclear pores during transport. (ie. List seven molecules that are involved in nuclear transport)
- RanGTP
- RanGDP
- GEF
- GAP
- Exportins
- Importins
- Karyopherins
- mRNA
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It is estimated that as many as 25% of kinetochore microtubules and 75% of interpolar microtubules are not anchored to the centrosome. In spite of that, all the microtubules are focused tightly at the spindle pole. Why do you suppose the microtubule ends that are not attached to centrosomes do not splay out away from the poles? Hint: Think about how minus- and plus-end directed motor proteins can move and what the motors might bind.
The negative ends of microtubules are focused at the spindle pore. Motor proteins bind to with the end of the kinetocore microtuble and the minus end of the interpolar microtubule. At the mitotic spindle, motor proteins bind with the plus ends of aniparallel microtubles.
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From these data, would you predict that cells treated with MI575 are arrested in the cell cycle at various stages (G1, S, G2, or M), or do you predict that cells will be blocked at a particular stage? If you think cells will be blocked at a particular stage, describe the stage at which you think the cells would be blocked.
I would predict that they would be arrested at metaphase.
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From the data, propose a simple molecular explanation for how MI575 acts to block the cell cycle.
MI575 inhibits APC which prevents the ubiquitination of M-cyclin. This is what prevents the cells from going into anaphase.
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Many features of __________________ cells make them suitable for biochemical studies of the cell cycle control system. The starting cells are unusually large and are arrested in __________________. When cells are triggered to resume cycling, by stimulation with the hormone __________________ they rearrest in __________________. Studies with Xenopus identified a partially purified activity called __________________ that drives a resting oocyte into M phase. MPF activity was found to be __________________ during the cell cycle and the amount of its kinase component, called __________________, remained constant. _________________ and _____________________of the kinase component of MPF regulates its activity and plays a role in its interactions with its partners, the __________________s. The components of MPF are evolutionarily __________________ from yeast to humans, so that the corresponding human genes are __________________ to function in yeast.
For each of the following sentences, fill in the blanks with the best word or phrase selected from the list below. Not all words or phrases will be used; each word or phrase should be used only once. (12points) Many features of frog egg cells make them suitable for biochemical studies of the cell cycle control system. The starting cells are unusually large and are arrested in G2. When cells are triggered to resume cycling, by stimulation with the hormone Progesterone they rearrest in Metaphase of meiosis II. Studies with Xenopus identified a partially purified activity called maturation promoting factor that drives a resting oocyte into M phase. MPF activity was found to be oscillatory during the cell cycle and the amount of its kinase component, called Cdk, remained constant Phosphorylation and Dephosphorylation of the kinase component of MPF regulates its activity and plays a role in its interactions with its partners, the cyclins. The components of MPF are evolutionarily conserved from yeast to humans, so that the corresponding human genes are able to function in yeast.
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Describe how G-Protein signaling works from the point where the ligand binds to the receptor to transcriptional activation. Be sure to explain what the GPCR (G protein coupled receptor) looks like.
- They consist of a single polypeptidechain that threads back and forth across the lipid bilayer seven times, forming acylindrical structure, often with a deep ligand-binding site at its center.
- Upon ligand-GPCR binding, the GPCR activated which in turn, binds to a trimeric G protein. This causes a confrontational of the AH domain of the alpha subunit the G proteins which releases GDP. A GTP then binds to that place which promotes the closure of the nucleotide-binding site. The alpha subunit separates from the receptor and betegamma complex. The alpha subunit and the betagamma complex then activate adenylyl cyclase which generates cAMP which activate PKAs.
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Name the complex of proteins that hold sister chromatids together ALONG THEIR LENGTH until they separate during mitosis.
Cohesins (SMC proteins)
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Describe how that complex is disrupted during late metaphase/early anaphase and results in sister chromatid separation. (ie. You need to name the component that disrupts this complex, what separates from it and how that results in sister separation for the onset of anaphase).
via ubiquitination of securin and separation from separase to cleave kleissi.Also there is a balance between CDK phosphorylates (activates) cohesins and PP2a dephosporylates cohesins. (unattaches).
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Lee Hartwell won the Nobel Prize in 2001 for his work with Saccharomyces cerevisiae and the cell cycle. This work involved generating yeast mutants (some of which were temperature sensitive) that were defective in various stages of the cell cycle. The mutants were called cell division cycle mutants, or cdc.
Why did Hartwell’s group have to generate temperature sensitive mutants? What kind of genes does this technique in particular, allow researchers to find?
TS mutants were generated because they allowed them to control the function of the proteins of the mutants. This allowed them to grow cells with mutants essential genes which are important for the survival of yeast.
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