The Nucleus

What's the maximum diameter for active transport vs. simple diffusion across the nuclear envelope?

Active transport: 26nm (NLS) Simple diffusion: 9nm

What happens when proteins are too large to diffuse through nuclear pores?

Actively transported; NLS enable protein to be recognized and transported by the nuclear pore complex

What is the job of the Nuclear-localization signal (NLS)?

to direct proteins to the nucleus (near the C-terminus of protein); usually 8-30 amino acids long; often contains proline and basic amino acids lysine and arginine

What transport problems are created by the nuclear envelope?

-All enzymes and other proteins required for chromosome replication and transcription of DNA in the nucleus must be imported from the cytosol -All the RNA molecules and partially assembled ribosomes needed for protein synthesis in the cytosol must be obtained from the nucleus

What are the general steps of protein targeting to the nucleus?

-All nuclear proteins are translated in the cytoplasm -Folded proteins are transported -Occurs via nuclear pore complexes

What did Alan Smith and colleagues discover about the NLS of the large T antigen?

-a stretch of 7 amino acids near the C-terminus of the protein is necessary for nuclear import; when only one of these seven amino acids, normally a lysine, is mutated to threonine, nuclear import of SV40 T antigen is blocked -SV40 T antigen NLS sufficient to drive a normally cytosolic protein, pyruvate kinase, into the nucleus

What effects does the relatively high concentration of Ran-GTP inside the nucleus have?

-nuclear Ran-GTP promotes the release of NLS-containing cargo from importin -nuclear Ran-GTP promotes the binding of NES-containing cargo to exportin

What is the structure of the Nuclear Pore Complex (NPC)?

-outer diameter of ~120nm -built from 30 different nucleoporins -octagonal symmetry -center granule (transporter) -circular cytoplasmic face, basket-like nuclear face

What are the steps of nuclear import via the Ran/Importin Pathway?

1. An NLS-containing cargo protein binds to importin 2. the importin-cargo complex is then transported through the NPC 3. Nuclear Ran-GTP binds to importin, triggering the release of the cargo protein in the nucleus 4. The Ran-GTP-importin complex is transported back to the cytosol, where 5. the hydrolysis of GTP to GDP is accompanied by the release of importin

True or False: Chromatin is located within the nucleus in a random fashion.

False: The chromatin of each chromosome has its own discrete location (chromosome territory)

Which substances are aqueous channels permeable to?

Ions and small molecules (including small proteins), because such substances quickly cross the nuclear envelope (the triphosphates required for DNA and RNA synthesis diffuse freely)

How is the Ran-GTP concentration maintained in the nucleus?

Maintained at high levels inside the nucleus by a guanine-nucleotide exchange factor (GEF) that promotes the binding of GTP to Ran in exchange for GDP

What was one of the first NLS sequences to be characterized?

NLS of the large T antigen, a protein produced by the simian virus 40 (SV40), discovered by Alan Smith

What is the nuclear envelope's inner and outer membranes separated by?

Perinuclear space

What recycling problem is posed by the Ran-dependent nuclear transport cycle? How is this solved?

Ran-GTP is exported along with molecules that leave the nucleus If Ran were not recycled back into the nucleus, it would be depleted: therefore, a specific nuclear transport protein NTF2 (Nuclear Transport Factor 2) shuttles Ran-GDP back into the nucleus

_____ provides structural support for the nucleus and attachment sites for chromatin.

The nuclear lamina

Small molecules moving through the nuclear pore complex experience a loss of entropy as they move through the pore. Which of the following is a likely strategy employed by the nuclear pore complex to overcome this loss of entropy of cargo?

The nuclear pore could form weak interactions with the cargo to decrease the enthalpy of the system. This is analogous to how an enzyme binds to a substrate and overcomes the loss of entropy of binding.

What is the nuclear lamina?

a thin dense meshwork of fibers lining the inner surface of the inner nuclear membrane; intermediate filaments lamins

What is the nuclear matrix (nucleoskeleton)?

an insoluble fibrous network that helps maintain the shape of the nucleus; chromosomes attach

The difference in direction between importin- and exportin-mediated transport is governed by _____.

interaction between Ran-GTP and these two classes of molecules, accompanied by a concentration gradient of Ran-GTP across the nuclear envelope

All __________ required by the nucleus must be transported in from the cytoplasm, and all __________ required for protein synthesis must be transported out to the cytoplasm.

proteins; RNA molecules

How is the Ran-GTP concentration maintined in the cytosol?

the cytosol contains a GTPase activating protein (GAP) that promotes the hydrolysis of GTP by Ran, thereby lowering the Ran-GTP concentration outside the nucleus

How can small particles diffuse freely back and forth through nuclear pores?

the pore complex contains tiny aqueous diffusion channels through which small particles and molecules can freely move; approximately 9nm in diameter, creates a permeability barrier for molecules significantly larger than ~30,000 in molecular weight

How is the direction of transport for any given cargo molecule determined?

the type of targeting sequence it contains: -Nuclear Localization Signal (NLS) for import -Nuclear Export Signals (NESs) for export