Nuclear Envelope & Transport

Nuclear Envelope Structure

• Consists of two concentric membranes, which are the inner and outer nuclear membranes.
• Includes the nuclear lamina, a fibrous network that provides structural support to the nucleus.
• Contains nuclear pore complexes, the only channels allowing molecules to travel between the nucleus and the cytoplasm.

Nuclear Lamina

• Supports the nuclear envelope, which is composed of lamins.
• The integrity of the nuclear lamina is regulated by phosphorylation/dephosphorylation.
• Lamin A/C mutation causes Hutchinson-Gilford Progeria syndrome.
• Lamin B mutation causes leukodystrophy (loss of myelin).
• Mutations in lamin binding protein emerin cause Emery-Dreifuss muscular dystrophy, characterized by stiff elbows, neck, heels, and heart problems.

Traffic Through Nuclear Pore Complexes (NPCs)

• Molecules can move through NPCs via passive diffusion or energy-dependent transport.
• Passive diffusion is used by small molecules.
• Energy-dependent transport is involves proteins and RNAs.
• DNA-binding proteins, histones, nonhistone proteins, activators and repressors of transcription, messenger RNA (mRNA)-binding proteins, components of the nucleus like lamins, ribosomal proteins, and shuttling nuclear transport receptors (importins) are transported via energy-dependent transport.
• mRNAs (together with mRNA-binding proteins), tRNAs (transfer RNAs), and 40S and 60S ribosomal subunits (complexes of ribosomal RNAs [rRNAs] and ribosomal proteins) move through NPCs.

Nuclear Pore Complex (NPC) Structure and Function

• NPCs are huge complexes, 15-30 times the mass of a ribosome, exhibiting octagonal symmetry.
• The channel of an NPC is 20-30 nm wide.
• FG (phenylalanine-glycine) domains form a hydrophobic sieve that blocks the diffusion of larger macromolecules, specifically those greater than approximately 40,000 Daltons.
• The model of a vertebrate nuclear pore complex (NPC) consists of a scaffold that anchors the complex to the nuclear envelope, a cytoplasmic and a nuclear ring, a nuclear basket, and eight cytoplasmic filaments.

Import of Proteins Through the NPC

• Proteins synthesized in the cytoplasm are targeted to the nucleus by a nuclear localization signal (NLS), such as P-K-K-K-R-K-V, which has basic residues.
• Proteins with an NLS bind to an NLS receptor, specifically importin α/β heterodimer.
• The protein/importin complex associates with cytoplasmic filaments.
• The protein/importin complex passes through the NPC.
• The complex associates with a GTPase called Ran.
• The Ran.GTP-importin β complex is transported back to the cytoplasm, where Ran is converted to Ran.GDP and brought back into the nucleus; importin α is returned to the cytoplasm via a protein named exportin.

GTP Binding and Hydrolysis

• GTP binding (activation) requires a protein called GEF (guanine nucleotide exchange factor).
• Hydrolysis of GTP to GDP (inactivation) requires a GAP (GTPase activating protein).

Nuclear Membrane Continuity

• The outer nuclear membrane is continuous with the rough endoplasmic reticulum (ER).
• The space between the inner and outer nuclear membranes is continuous with the lumen of the rough ER.

Ribosome Structure

• Eukaryotic (vertebrate) ribosomes have 28S, 5.8S, and 5S rRNA subunits totaling 50 proteins, and 18S rRNA subunits totaling 33 proteins.
• These subunits assemble into 80S ribosomes.
• Prokaryotic ribosomes have 23S and 5S rRNA subunits totaling 31 proteins, and 16S rRNA subunits totaling 21 proteins.
• These subunits combine into 70S ribosomes.

Early Origins of the Nucleus

• An ancient prokaryotic cell, containing DNA and membrane-bound ribosomes, evolves to contain a endoplasmic reticulum, inner and outer nuclear membrane, a nuclear pore, a nucleus and the cytsosol to become an ancient eukaryotic cell.

Nucleolus

• The nucleolus is a suborganelle of the nucleus.
• It functions as a site where ribosomes are assembled.

Ribosome Assembly in the Nucleolus

• Ribosomal proteins are imported to the nucleus from the cytoplasm and then delivered to the nucleolus.
• These proteins assemble on pre-rRNA (pre-ribosomal RNA).
• The pre-rRNA is cleaved to form several rRNAs.
• Ribosomal proteins and rRNAs assemble to form the 40S and 60S ribosomal subunits.
• These subunits are exported to the cytoplasm because the fully assembled ribosome is too large to fit through the pore, ensuring protein synthesis takes place in cytoplasm.

Chromatin Structure

• Chromosomal DNA is packed into chromatin fibers with the aid of specialized proteins.
• Complexes of eukaryotic DNA and proteins are called chromatin.
• Chromatin contains about twice as much protein as DNA.
• The major proteins of chromatin are histones.
• Histones are small proteins (11 to 23 kDa) containing a high proportion of basic amino acids (arginine and lysine), facilitating binding to the negatively charged DNA molecule.
• The 5 major types of histones are H1, H2A, H2B, H3, and H4, which are very similar among different species of eukaryotes.
• Chromatin also contains an approximately equal mass of nonhistone chromosomal proteins, with more than 1000 different types.

Chromatin Organization

• DNA is wrapped around an octamer of histones H2A, H2B, H3, and H4 in a nucleosome core particle and sealed by histone H1.
• This produces a 7-fold compaction of the DNA.
• Non-histone proteins bind to the linker DNA between nucleosome core particles.
• Formation of 30 nm fibers is dependent upon histone interactions and further increases compaction by approximately 6-fold.
• The 30 nm fibers are then organized into 80-100 nm supercoiled loops, possibly stabilized by a protein called cohesin.
• Mitotic chromosomes represent the ultimate in chromatin compactness, with a ratio of 10,000:1 (1 μm of mitotic chromosome length contains approximately 1 cm of non-compacted DNA).
• Chromatin condenses into chromosomes that can be distinguished in the light microscope as a cell prepares to divide into two daughter cells.
• The chromosomes can be arranged in a karyotype, a preparation of homologous pairs ordered according to size, and may be used to screen chromosomal abnormalities.

Heterochromatin vs Euchromatin

• Euchromatin returns to a dispersed state after mitosis.
• Heterochromatin is condensed during interphase.
• Constitutive heterochromatin remains condensed all the time and is found mostly around centromeres and telomeres; it consists of highly repeated sequences and few genes.
• Facultative heterochromatin is inactivated during certain phases of an organism's life, such as X-inactivation.