Transport of RNAs and proteins across nuclear envelope Flashcards
What are important parameters for phase seperation (LLPS)? What can it be used for?
(1)
- proteins contain IDRs, protein concentration, PTMs of proteins.
- temp, pH, ionic strength.
- presence of binding partners: proteins, RNA, DNA.
(2)
- can regulate enzyme activity by sequestering an enzyme in an inactive state until specific conditions are met. can ionize an enzyme within a phase-separated compartment where it becomes active.
- inactivation, activation, buffering, force generation, filtration, localization, sensing.
- mRNA retention in nucleus during glucose stress, tight junctions, DNA repair etc.
- treatment resistance, cancer, infectious diseases.
What are advantages of biocondensates compared to organelles?
- no energy/recourses requires to generate membranes, materials can move freely in and out of due to lack of membrane barrier.
- their formation and disassembly can be easily controlled, allowing for rapid adaption to cellular needs.
- highly dynamic and quickly regulated.
- assembled when needed, disassembled when not.
What is the function of FXF repeats in the central gates channel at the NPC?
- generates transport selectivity
- help control transport between nucleus and cytoplasm, acting as a selective barrier.
Can be described as:
(1) Gummy Bear Model: This model often refers to a flexible, elastic, or soft structure. In terms of protein structure, this could be used metaphorically to describe proteins or sequences that have a flexible or disordered conformation, which might be the case for certain FXF repeats in specific proteins.
- gel-like structures, allowing selective passage of specific molecules.
(2) Oily Spaghetti Model: This is a model used to describe membrane-associated proteins or structures with a hydrophobic core, often like the way proteins interact with lipid membranes. If FXF repeats are part of a protein that interacts with the membrane, this could be a suitable metaphor, especially if those repeats influence the protein’s hydrophobicity or its interaction with lipid environments.
- repeats behave like disordered, hydrophobic strands, creating a tangles but dynamic mesh.
FG- nucleoporins
- contain FG repeats (undergo LLPs - form droplet like structures, help create NPCs selective permeability)
- crucial for NPC function
- LLPS allows it to form flexible, dynamic barrier in nuclear pore.
- interact with nuclear transport receptors, guiding molecules across NPC.
- prevent random diffusion of large proteins and RNA between nucleus and cytoplasm.
- hydrophobic nature helps with molecule recognition and binding to transport receptors
- FXF repeats and FG nucleoporins ensure only right molecules get in and out of nucleus.
What are the two ways of movement across the NPC?
(1) Passive Diffusion
- molecules have to be smaller than diffusion channel of the NPC (diameter around 8nm), inefficient for proteins > 40 kD.
- proteins > 70 kD are excluded from nucleus if they have no import signal.
- no energy.
- no transport apparatus needed.
- movement depends on concentration gradient of the molecule.
(2) Active Diffusion
- N -> C or C=-: N
- specialized transport apparatus (soluble factors and nucleoproteins)
- molecules that exceed diffusion radius of NPC, particles up to 45 nm can be translocated if they have proper signal.
What signals control the nucleocytoplasmic distribution of proteins?
(1) Signals for Transport.
- NLS (nuclear localization sequence), C-> N
- NES (nuclear export signal), N-> C
- shuttling signals/specific for each sequence or one that combines import/export.
(2) Signals for retention.
- NRS (nucleus retention signal) (nucleus)
- CRS (cytoplasmic retention signal) (cytoplasm)
NLS
- C -> N
- Sr 40 tppKKKRKr (classical)
-> contains (+) amino acids (K & L), both charge and structure crucial for nuclear import. if mutated, protein stays in the cytoplasm. - nucleoplasmin avKRp…KKK
- permanent signals (not cleaved)
- can be located anywhere within the polypeptide chain
- mediate post translational nuclear import
- non-classical: stretches of aa residues with pY-motif. SR-rich regions (RNA binding proteins)
- if ionophore, increase Ca2+, enhancing nuclear import, when removed, stay in cytoplasm.
What is the model for classical important into the nucleus?
(1) Recognition & binding.
- protein with NLS (importin) recognized by NLS receptor (2 subunits, alpha subunit that binds to NLS on cargo protein, and beta subunit that interacts with nuclear transport machinery)
(2) Docking at NPC.
- importin-cargo complex dock at NPC by binding to cytoplasmic filaments.
- no energy needed.
- occurs at 4C.
(3) Translocation into the Nucleus
- cargo-receptor complex moves through NPC into nucleus.
- needs energy.
- does not occur at 4C.
(4) Cargo release & recycling.
- inside nucleus, GTP-bound small GTPase (RanGTP) binds beta-subunit, triggering cargo release.
- import receptors recycled back to cytoplasm for reuse.
- requires energy.
How does HIV-I go through nuclear import without transport receptors?
- viruses like HIV-I face challenges entering nucleus cause NPC restricts large molecules from passing through (too large to use normal nuclear transport receptors)
- instead, they directly interact with FG-nucleoporins (proteins that line NPC).
- ‘capsid’ melts into FG phase, to slip through NPC.
- allows HIV-I to enter nucleus without exposing its genome to antiviral defenses in the cytoplasm.
How do we achieve directionality in nuclear trafficking?
- RanGTP/GDP gradient controls the directionality of nuclear trafficking.
- RanGTP is found in the nucleus, and RanGDP is found in the cytoplasm, creating a gradient.
- RCC1, bound to chromatin in the nucleus, converts RanGDP into RanGTP. This ensures high levels of RanGTP in the nucleus and low levels in the cytoplasm.
- In the cytoplasm, RanGAP converts RanGTP into RanGDP, maintaining the gradient.
- Nuclear import: RanGTP in the nucleus binds to importins, releasing the cargo.
- Nuclear export: RanGTP binds to export receptors, driving the export of cargo.
What are the factors for nuclear protein import?
(1) Transporters (nuclear carriers)
- importin-B: bind to RanGTP.
- exportin.
- transportin.
-karyopherin.
(2) Adaptors
- importin - alpha (helps importin beta)
- not always needed for cargo binding
(3) Ran-GTPase - controls directionality.
(4) Ran-interacting factors
- RanGAP (GTP->GDP)
- RCC1 (RanGEF) (GDP->GTP)
- RanBPI, RanBP3 - help regulate Ran function.
(5) Nucleoporins (Nups)
- form NPC.
- certain FXF repeats help interact with transporters.
(6) Transport signals
Nuclear Transport & Aging (young nuclei vs old nuclei)
(1) Young Nuclei:
- interact with NPC, maintains strong permeability barrier.
(2) Old nuclei:
- central gated channel becomes leaky (in NPC) over time.
- NPC function deteriorates, increasing permeability.
- small molecules still diffuse normally, but large molecules that were previously included can now passively enter.
- permeability barrier weakens, reducing nuclear transport efficiency.
What are the consequences when NPC permeability breaks down?
- loss of compartmentalization (uncontrolled movement between nucleus and cytoplasm)
- gene regulation disruption (unwanted molecules in nucleus may interfere with transcription and chromatin organization)
- mislocalization of RNA and ribosomal subunits
- increase disease risk (aging and neurodegenerative disorders)
What are the levels of nuclear trafficking control?
(1) Individual Protein/Molecule level
- modifying NLS
(2) NLS receptor concentration
- increase NLS receptors, increase nuclear import.
(3) NPC level
- adjusting central gates channel/diameter affects which molecules can enter/leave the nucleus.
For herpesvirus, what is an alternative route of nuclear import?
- too large
- capsid interacts with INM forming temporary envelope (similar to endocytosis)
- capsid can now bud into the perinuclear space, moving from INM to ONM.
- envelope then removed (de-envelopment) releasing capsid into cytoplasm.
- enables large cargos to exit nucleus without NPC.
