NPC Transport Flashcards
Nucleocytoplasmic transport requires
Energy
Protein receptors
Unique targeting signals
Nuclear Localization Signals
Amino acid sequence that is both necessary and sufficient for cytoplasm to nuclear targeting
Recognized by nuclear receptor proteins
Classic NLS
Most common
Consists of a short stretch of positively charged (basic) amino acid residues
KKQRKKx
X= S/T/Y (can be phosphorylated)
Bipartite NLS
Composed of 2 short stretches of basic amino acids and a 7-19 amino acid long “spacer” sequence
-2 sequences with a linker
Transport Receptors
Mobile protein responsible for moving “ferrying” protein “cargo” across the nuclear envelope
Karyoferins
Large family of receptor proteins (transport receptors) responsible for moving macromolecules either into nucleus (importins) or out of nucleus (exportins)
ARC1
Shuttle between cytoplasm and nucleus
Posses a classic NLS (aa 261-266)
Mutation of ARC 1 NLS
Mutation of residues 261-266 results in in myc-tagged ARC1 being mislocalized exclusively to the cytoplasm
Myc- short polypeptide derived from the c-myc oncogene protein
- Acts as a tag for immunodetection
Addition of ARC1 NLS
Fusion of residues 261-266 in ARC1 to the cytoplasmic protein CAT results in the fusion protein (CAT-NLS) being redirected to the nucleus
Ran-GAP1
Hydrolyzes GTP to GDP on Ran-GTP in cytoplasm
RCC1
Converts Ran GDP to Ran-GTP in nucleus
NES
LxxLxxL
X= any amino acid
Nucelocytoplasmic Transport of ARC1:
Before pollination- localized in nucleus
During self pollination
- NLS is disrupted due to phosphorylation of adjacent amino acid residues
- NES> NLS(p)- localized in cytoplasm
- Functions in the ubiquitin-mediated turnover of other proteins, leading to pollen rejection (no fertilization)
Mutation of NES results in ARC1 being mislocalized to the nucleus
Cell cycle checkpoints
Mid G1 (Cell commits to DNA replication and organelle duplication in S phase)
End of G2- Cell commits to enter Mitosis
End of metaphase- cell commits to chromosome segregation
Cyclins
Transition through checkpoints is controlled by cyclins and CDKs
CDKs phosphorylate target nuclear proteins when bound to cyclin
Concentration varies in a cyclic fashion during cell cycle (synthesized and degraded during each cell cycle)
Maturation Promoting Factor (MPF)
□ Heterodimeric protein consisting of a kinase and regulatory cyclin subunit
Critical for G2/M transition during cell cycle
Phosphorylation of histones and condensins
Phosphorylation leads to chromatin packing and chromosome condensation
Phosphorylation of lamins
Phosphorylation leads to disassembly of the nuclear lamina
Phosphorylation of nups
Phosphorylation leads to disassembly of NPC
Open Mitosis
Dynamic process in higher eukaryotes
- Lower eukaryotes exhibit "closed mitosis"
- Nuclear envelope remains intact
Entire nucleus disassembles by metaphase
-Nuclear outer and inner membranes fragment, lamina and NPCs disassemble, all soluble nuclear proteins are released into cytoplasm
2 daughter nuclei reassemble during telophase (after chromosomal division)
Low [cyclin] = low CDK activity = dephosphorylation of nups and lamins= reformation of nuclear lamina, envelope, and NPC re-import of soluble NLS-proteins from the cytoplasm
Binding of Different Cyclins
Different cyclins can bind to the same CDK (Cyclins cause a conformational change that activates the CDK)
Cdc2
CDK that controls the transition from G1 to S (START) and G2 to M phase by binding to different cyclins
End of G1
-High [G1 cyclins] = High cdc2 activity= cell enters S phase
End of G2
-High [mitotic cyclins] = high cdc2 activity = cell enters M phase
Cyclin Oscillation
Oscillation in different cyclin concentration during the cell cycle leads to changes in CDK activity
Oscillation due to rates of protein synthesis and degradation at different points of the cell cycle
Decrease in [cyclin] after the start of S and M phase due to decreased synthesis of new cyclin proteins and degradation of pre-existing cyclin proteins
CDK Inactivation
CDKs themselves are inactivated by phosphorylation from CDK kinases
Pre-existing cyclins also prevented from targeting to the nucleus
-Can’t activate CDKs in nucleus
