the nucleus Flashcards
2 main functions of the nucleus
- compartmentalization of the cellular genome and its activities
- DNA replication, transcription, RNA processing - coordination of cellular events
- control of metabolism, protein synthesis, reproduction
what components make up the nuclear envelope
- nuclear membrane
- nuclear lamina
- nuclear pores
what does nuclear content consist of (inside)
- chromatin
- nucleoplasm
- nucleolus
nuclear envelope: structure
- 2 parallel phospholipid bilayers
- outer membrane binds ribosomes and is continuous with ER
- inner membrane has integral membrane proteins that connect it to the nuclear lamina
- inter membrane space is continuous with the ER lumen
- inner and outer membranes join at nuclear pores
nuclear envelope: functions
- separates nuclear content from the cytoplasm
- selective barrier: passage of molecules from nucleus to cytoplasm, regulates gene expression
- binds nuclear lamina (structural framework)
What is the nuclear lamina
- thin meshwork bound to the inner membrane of the nuclear envelope
- includes A, B and C lamins
- supports structure of the nuclear envelope
- scaffold for chromatin attachment
nuclear content: chromosomes
- location of a gene is related to its activity (active genes found at periphery of a chromosomal subdomain)
- inter-chromosomal channels are regions between domains as barriers between DNA-DNA or DNA-protein interactions
- active genes (chromatin) from different subdomains extend to form transcription factories
Nuclear speckles
- subdomains where mRNA splicing factories are concentrated
- often located next to transcription factories
the nucleolus
- NOT membrane-bound
- site of ribosome biogenesis
- initial stages or ribosomal subunit assembly (rRNA + protein)
nuclear pores
- gateways between the cytoplasm and nucleoplasm
- inner and outer membranes of the nuclear envelope fuse at pores
- not evenly distributed across the membrane
Nuclear pore complex
protein structure that fills the nuclear pore
- fits into the pore and reduces the functional diameter
- extends to the cytoplasm and nucleoplasm
NPC structure
- octagonal symmetry
- structural and membrane nucleoporins
- FG domains
- cytoplasmic filaments
- nuclear basket
structural and membrane nucleoporins
- anchors the NPC to the nuclear envelope
- forms an aqueous central channel
- inner surface of the channel is lined by FG NUPS
- located on the cytoplasmic and nuclear side of the NPC
- link the central scaffold and cytoplasmic filaments or nuclear basket
FG Nups
- FG nucleoporins have large FG amino acid repeats
- FG domains possess highly disordered secondary protein structure
- FG domains extend into the central channel
- form a hydrophobic mesh which limits diffusion of macromolecules
cytoplasmic filaments
- extend into the cytosol on the cytosolic side of the NPC
- involved in nuclear receptor-cargo protein recognition and import
nuclear basket
- located on the nuclear side of the NPC
- involved in nuclear receptor-cargo protein import and export
functions of the NPC
- passive diffusion of small molecules across the NPC
- regulates the movement of larger molecules
1. import of nuclear proteins and RNAs into the nucleus
2. export of RNAs, ribosomal subunits and proteins from the nucleus
what is nucleoplasmin
nuclear protein
- synthesized in the cytoplasm, associates with cytoplasmic filaments and translocates into the nucleus
Nuclear localization signal (NLS)
- used for targeting in cytosol-to-nucleus transport
- specific sequence of AAs that are recognized by nuclear receptor proteins
how is an NLS both necessary and sufficient for cytosol-to-nucleus targeting
Necessary: if it is mutated/not present, the protein will fail to target the nucleus
sufficient: if the sequence linked to a passenger protein is capable of redirecting the resulting fusion protein to the nucleus
classic vs bipartite NLS
classic: short stretch of positive AA residues
bipartite: 2 short stretches of basic AAs and a 7-10 AA long spacer sequence
Karyopherins
- mobile proteins responsible for moving protein”cargo” across the nuclear envelope
- large family of receptors responsible for moving macromolecules
importins: move into the nucleus
exportins: move out of the nucleus
what is Ran protien
- small G-protein involved in nuclear import and export
- serve as molecular switches in the transport process
- Ran-GTP = active
- Ran-GDP = inactive
- GTP hydrolysis provides the energy required for nucleocytoplasmic transport
consequences of nuclear import failure
- mutation in the NPC would lead to immediate cell death
- mutation in the NLS may lead to disease (e.g. Sawyer syndrome)
nuclear import of proteins: step 1
the NLS-containing cargo protein is recognized in the cytosol by importin-alpha
- then binds importin-beta to create a heterodimeric complex
nuclear import of proteins step 2
the protein-importin receptor complex moves through the cytosol toward the nucleus via the cytoskeleton
- at the surface of the nucleus importin-beta binds to a cytoplasmic filament at the NPC
nuclear import of proteins: step 3
the protein-importin receptor complex is translocated through the NPC
- protein-receptor complex interacts with FG domains of the FG Nups, this dissolves the FG-domain network to allow for translocation through the channel
nuclear import of proteins: step 4
cargo-receptor complex associates with the nuclear basket and binds to Ran-GTP (via importin-B) resulting in its release from the NPC
- the complex disassembles in the nucleoplasm
nuclear import of proteins: step 5
Ran-GTP bound importin-B moves back to the cytosol due to [Ran-GTP] gradient
- in the cytosol GTP on Ran-GTP is hydrolyzed by Ran-GAP1, and Ran-GDP is released from importin-B
Ran gradient in the cell
[Ran-GTP] nucleus > [Ran-GTP] cytosol
[Ran-GDP] nucleus < [Ran-GDP] cytosol
Ran accessory proteins
GAP = Ran-GAP1: cytosolic protein, promotes hydrolysis of Ran-GTP to Ran-GDP
GEF = RCC1: nuclear protein, promotes conversion of Ran-GDP to Ran-GTP
nuclear export signal (NES)
specific AA sequence that is recognized by exportin to mediate targeting of the protein from the nucleus to the cytosol
- most common NES consists of a leucine-based motif
what is the fate of importin-alpha in the nucleus?
- rely on export signals (NES) to get back to the cytoplasm so it can be used for nuclear import again
- gets out the same way ‘cargo’ proteins do
Nuclear export of proteins: step 1
importin-alpha (or any other cargo protein) binds to exportin via its nuclear export signal (NES)
Nuclear export of proteins: step 2
the importin-alpha-exportin complex binds Ran-GTP in the nucleus
- Ran-GTP promotes stable assembly of the importing-alpha-exportin complex
nuclear export of proteins: step 3
importin alpha-exportin-Ran-GTP complex is transported via the NPC to the cytosol
- following the [Ran-GTP] gradient
nuclear export of proteins: step 4
in the cytosol, GTP on Ran-GTP is hydrolyzed by Ran-GAP1
nuclear export of proteins: step 5
Ran-GDP is released from exportin which causes the release of importin-alpha (or the NES containing cargo)
- exportin moves back to the nucleus (via importin) for another round of export
Nuclear export of mRNA: step 1 (Ran-independent)
multiple dimers of NXF1 and NXT1 bind to processed mRNA - the NXF1/NXT1 dimer direct the RNA/protein complex to the NPC channel
Nuclear export of mRNA: step 2 (Ran-independent)
NXF1/NXT1 transiently interact with FG-domains in FG Nups
Nuclear export of mRNA: step 3 (Ran-independent)
as the mRNA moves through the NPC, an RNA helicase removes the NXF/NXT dimer in an ATP-dependent manner
