Import and Export of the Nucleus Flashcards
What types of compounds are imported and exported from the nucleus?
How is p53 expression affected by nuclear import or export in carcinogenesis?
p53 may be exported from the nucleus in cancer cells, mislocalizing in the cytoplasm, and thus causing the breakdown of the DNA damage checkpoints it is involved in.
What is the basic structure and thickness of the nuclear pore complex (3 layers)?
Membrane Layer - Anchored into the nuclear envelop
Scaffold Layer - Links the Membrane and Barrier layers and provides curvature to the structure.
Barrier Layer - Performs the function of acting as a selective gate.
Entire pore is ~200nm thick
How many proteins are contained within one nuclear pore complex, how long do these proteins persist?
Between 500 and 1000 proteins (from 30-50 different types) are combined to form one nucelar pore complex. Proteins in the scaffold layer may persist for days, weeks, or years in their position. Proteins in the barrier layer may only remain in place for seconds or minutes, and many have roles outside of the pore complex (including in gene regulation).
What are the two primary structure types resulting from FG repeats, and what is their function
FG = Phenylalanine Glycine
The to strucutre types are cohesive (GLFG) that bind to themselves, and non-cohesive (FXFG) that do not bind to themselves. They are differentially distributed throughout the barrier layer to creat different domains that increase the efficiency of trafficing through the pore. They provide a high rate of low-affinity interactions that facilitate movement through the pores without direct energy input.
Because the barrier proteins often move molecules via brownian motion and low-affinity bonding, why is direct energy needed and how is it used in import/export?
Direct energy is used to determine the directionality of the movement through the pore, ie. it determines whether a compount is imported or exported. (Likely through concentration gradients?)
What proportion of barrier proteins contain FG repeats, how many binding sites does this create, and how do they bind molecules?
Approximately 30% of all barrier proteins have at least 4 FG repeats, totalling up to ~3500 binding sites within each pore, and creating 12% of the total mass of the pore. B/c FG repeats are non-polar, they bond with hydrophobic regions of target molecules.
What is a major difference between the function of nuclear pore complexes and ion channels?
Nuclear pores are always open and thus electrically neutral. They allow the free flow of H2O, ions, ATP and sugars. They still present a physical barrier to hydrophilic molecules larger than 50kDa.
What simple principle does the nuclear pore complex rely on to maintain its function?
The central “channel” of the NPC is essentially a hydrophobic space, maintained by the interactions of the FG repeat sections in the barrier layer proteins.
What are the three basic types of movement through a nuclear pore complex?
Size filtering diffussion - Movement of small, hydrophilic molecules through the pore (ions, sugars, etc.)
Spontaneous migration - Where larger hydrophilic molecules (B-Catenin, or karyopherins) shift their conformation and expose hydrophobic regions to interact with FG repeats.
Facilitated Transport - Where large, hydrophilic molecules interact with transport molecules to cross the pore.
What is the major difference between facilitated transport and spontaneous migration or diffusion, and what molecule is required?
Unlike the other methods, facilitated transport can move compounds against their concentration gradient. It requires the target molecule to bind with karyopherin to acheive transit through the NPC, and the transit often occurs through the edges of the pore, not through the central channel. This also requires the input of energy.
What are four primary features of facilitated transport?
Energy Dependent
Temperature Dependent
Signal Dependent
Saturable
What are the two categories of Karyopherin proteins?
Receptor Family - Bind with RanGTP and Adaptor Karyopherins and/or directly to cargo, also interact directly with FG repeats in barrier proteins. Different affinities between Receptors and FG repeats aid in efficiency.
Adaptors - Cargo selective binding, binds directly to cargo and forms linkage to Receptors for transit. Cannot bind to RanGTP or interact with FG domains.
What differentiates a protein moving across an NPC from one being made on the ER, and what does a protein need to move through the NPC?
Proteins are fully folded at all times during the transit across the NPC. Proteins may have a NLS, an NES, both NLS and NES, or neither and be bound to another moletule that does have them.
What is the specifc sequence of events for facilitated transport of cargo into the nucleus?
The Adaptor binds to the cargo and the Receptor, forming an import competent complex that is tranported into the nucleus. Inside the nucleus, the RanGTP binds to the complex and breaks the Adaptor and Receptor apart, releasing the cargo.
What drives the Ran cycle?
The primary driver of the Ran cycle is the high relative concetration of RanGTP inside the nucleus. RanGTP flows down its concetration gradient by exiting the cell, where it is hydrolyzed by RanGAP (an activating protein). RanGDP is then returned down its concentration gradient into the nucleus via NTF2. Reversing these concentrations drives the cycle backwards. Ran comprises 0.4% of all cellular contents, 10^7 copies/cell.
One example of a mutation affecting protein-protein interactions leading to pathogenic disruption of nuclear import/export
Rad51 binds to BRCA2 in a manner that hides its NES. At times of DNA damage, the BRCA2/Rad51 complex is imported into the nucleus and DSS1 covers BRCA2’s NES, allowing it to assist in checkpoint controlls. If a mutation occurs that inhibits Rad51 from binding to BRCA2, both NES are exposed and both are exported, leading to the increased genomic instability.
