Lecture 4 - nucleus part 3 Flashcards
What is piggyback nuclear protein import?
some proteins being imported into the nucleus without an NLS
- newly synthesized protein lacking NLS binds to NLS-containing protein in cytoplasm
What determines the distribution of protein in either the cytoplasm or the nucleus?
strength of the NLS or NES
What is the strength of the NLS or NES controlled by?
post translational modifications
What does ARC1 possess?
both NLS and NES
What occurs before and during pollination in ARC1 in regard to NLS and NES?
before pollination - NLS>NES results in ARC1 being localized mostly in nucleus
during self pollination - NLS disrupted due to phosphorylation of adjacent AA residues
NLSphos < NES results in ARC1 localized mostly in cytoplasm, ARC1 in cytoplasm functions in proteosome dependent turnover of proteins
What are two experiments used to determine the nucleocytoplasmic transport of ARC1?
experiment 1: phosphorylation of NLS results in ARC1 mislocalized to only cytoplasm. Introduced phosphomimic mutations to residues adjacent to NLS - aspartic acid physiochemically similar to phosphorylated S/T/Y
experiment 2: mutation of NES results in ARC1 mislocalized to only nucleus
What are the steps to an in vitro coIP assay to assess cargo protein importin binding?
(does a nuclear localized protein interact with importin?)
1. mix bait and or prey proteins in vitro
2. add agarose beads coated with anti-epitope tag IgGs
3. isolate beads via centrifugation along with all associated proteins
4. SDS-PAGE and coomassie blue staining
What are the two main components to a CoIP assay?
bait - purified epitope tagged nuclear protein
prey - purified importin (alpha beta subunits)
What are the two main stages to the cell cycle?
interphase and m phase (mitosis)
What are the three(four) stages in interphase?
G1 - Gap 1 cell performs normal cellular activities and can respond to the environment
S - synthesis DNA replication and increased synthesis of factors required for chromosome duplication
G2 - gap 2 cell grows and prepares for mitosis
G0 - gap 0 non-dividning cells, stage when a cell arrests during G1 rather than proceeding to S
What occurs in M phase (mitosis)?
prophase, metaphase, anaphase, telophase, cytokinesis (mother cell divides into two daughter cells)
What regulates cell cycle progression or arrest?
by checkpoints, involve surveillance mechanisms to ensure cell cycle proceeds properly, if not cellular signals can lead to death, or cancer
Where are the checkpoints in the cell cycle?
mid G1 - START cell commits to DNA replication in S and organelle duplication begins
end of G2 - cell commits to entering M phase
end of metaphase - cell commits to chromosome segregation
What are nucleocytoplasmic mobile factors?
responsible for mediating transitions from one phase to the next in the cell cycle
What is transition through checkpoints in cell cycle controlled by?
mitotic cycles an cycle-dependent kinases (CDKs)
What are CDKs?
cell-specific kinase enzymes located in nucleus; phosphorylate various target nuclear proteins - turn on or off
What are cyclins?
nucleocytoplasmic proteins; bind CDKs and regulate their activity during specific stages of the cell cycle
How do cyclin and CDK activity vary throughout the cell cycle?
early interphase (G1) - [mitotic cyclins] low -> CDK activity low
end of G2/start of M phase - [mitotic cyclins] high -> CDK activity high
What are three examples of CDK target proteins in the nucleus at the end of G2 and start of M phase?
histones/condensins - phosphorylation leads to chromatin packing and chromosome condensation in prep for mitosos
lamins - phosphorylation leads to disassembly of nuclear lamina
nups - phosphorylation leads to disassembly of NPCs
What occurs to the nucleus during mitosis?
nucleus completely disassembled by metaphase
prophase - outer and inner nuclear membranes break down, lamina and NPCs disassemble, membrane-bound and soluble nuclear proteins released into ER membrane and cytoplasm respectively
two daughter nuclei reassemble during end of mitosis
What are the steps to mitosis in the nucleus and activity of CDK and cyclin?
[mitotic cyclin] low -> CDK activity low -> dephosphorylation of nups and lamins -> reformation of nuclear lamina, nuclear envelope and NPCs -> re import of soluble NLS proteins from the cytoplasm
Why are there oscillations in [cyclin]?
relative rates of protein synth and degradation at diff points in cell cycle
why is there a decrease in [mitotic cycle] after the start of M phase?
due to decrease synth of new cyclin proteins and degradation pre-exisitng cycle proteins via proteosome
What are CDKs inactivated by?
phosphorylation by CDK kinases, CDK kinase kinases etc.
