16.3 Nuclear import and export

Nuclear Pore Complex and Nuclear Transport

The nuclear pore complex controls what gets in and out of the nucleus. Small molecules diffuse through freely, but larger proteins and RNA need specific signals and transport machinery to cross. This selective gating is essential for gene expression, since transcription happens in the nucleus but translation happens in the cytoplasm.

Structure of the Nuclear Pore Complex

The nuclear pore complex (NPC) is a massive, multiprotein channel embedded in the nuclear envelope. Each NPC is built from proteins called nucleoporins (Nups), arranged with 8-fold rotational (octagonal) symmetry around a central channel. This channel connects the cytoplasm to the nucleoplasm and supports transport in both directions.

The NPC acts as a selective gateway:

• Small molecules (ions, metabolites, and proteins under ~40 kDa) pass through by passive diffusion.
• Larger macromolecules (most proteins and RNA) require active, signal-dependent transport. They can't just slip through; they need specific targeting signals and dedicated transport factors to make the trip.

Role of Nuclear Localization Signals

A nuclear localization signal (NLS) is a short amino acid sequence on a protein that flags it for import into the nucleus. Classical NLS sequences are rich in the basic (positively charged) amino acids lysine and arginine. They come in two flavors:

• Monopartite NLS: a single stretch of basic residues
• Bipartite NLS: two stretches of basic residues separated by a linker

The NLS doesn't get cleaved off after import. It stays on the protein, which means the protein can be re-imported after mitosis when the nuclear envelope reforms.

Recognition works through a two-part receptor system:

1. Importin-α acts as an adaptor. It directly recognizes and binds the NLS on the cargo protein.
2. Importin-β binds to importin-α and mediates the actual translocation through the NPC by interacting with nucleoporins.

So the chain is: cargo → importin-α → importin-β → NPC.

Mechanism of Ran-Dependent Transport

The small GTPase Ran provides directionality to nuclear transport. Ran exists in two nucleotide-bound states, and their distribution across the nuclear envelope is asymmetric:

• Ran-GTP is concentrated in the nucleus, maintained by RCC1 (a guanine nucleotide exchange factor that loads GTP onto Ran).
• Ran-GDP is concentrated in the cytoplasm, maintained by RanGAP (a GTPase-activating protein that stimulates Ran to hydrolyze its GTP).

This steep Ran-GTP/Ran-GDP gradient across the nuclear envelope is what drives the directionality of both import and export.

Nuclear Import (step by step):

1. In the cytoplasm, the importin-α/importin-β complex binds the NLS-containing cargo.
2. The entire complex translocates through the NPC.
3. In the nucleus, Ran-GTP binds importin-β, causing a conformational change that releases the cargo.
4. Importin-β (bound to Ran-GTP) and importin-α are recycled separately back to the cytoplasm. Once there, RanGAP triggers GTP hydrolysis on Ran, releasing importin-β so it can participate in another round of import.

Nuclear Export (step by step):

1. In the nucleus, an exportin (such as CRM1) binds both a cargo protein bearing a nuclear export signal (NES) and Ran-GTP. All three components are needed to form a stable export complex.
2. The exportin-cargo-Ran-GTP complex translocates through the NPC.
3. In the cytoplasm, RanGAP stimulates hydrolysis of Ran-GTP to Ran-GDP. This destabilizes the complex, releasing the cargo.
4. The exportin and Ran-GDP are recycled back to the nucleus, where RCC1 recharges Ran with GTP.

The key idea: Ran-GTP promotes cargo release from importins (in the nucleus) but promotes cargo binding to exportins (also in the nucleus). This asymmetry is what makes the system work.

Import vs. Export of Macromolecules

Protein Import:

• Mediated by importins (importin-α/β) that recognize NLS sequences
• Cargo is released in the nucleus when Ran-GTP binds importin-β
• Driven by the Ran-GTP/GDP gradient

Protein Export:

• Mediated by exportins (e.g., CRM1) that recognize NES sequences
• Export complex formation requires Ran-GTP, so it only assembles in the nucleus
• Complex disassembles in the cytoplasm upon GTP hydrolysis

RNA Export:

RNA export follows different rules depending on the RNA type:

• mRNA export is tightly coupled to processing. Only fully spliced, capped, and polyadenylated mRNAs are exported, which serves as a quality control step. Mature mRNAs travel as messenger ribonucleoprotein particles (mRNPs), and their export is mediated by the NXF1-NXT1 heterodimer (also called TAP-p15) along with adaptor proteins like the TREX complex. Notably, mRNA export is not Ran-GTP dependent.
• tRNA is exported by exportin-t in a Ran-GTP-dependent manner.
• pre-miRNA is exported by exportin-5, also Ran-GTP dependent.
• rRNA subunits are exported as pre-ribosomal particles through their own dedicated pathways.

The fact that mRNA export uses a distinct, Ran-independent mechanism (NXF1-NXT1) while most other RNAs rely on Ran-dependent exportins is a common exam distinction worth remembering.