Ch 9-The Nuclear Envelope and Traffic between the Nucleus and the Cytoplasm

In Animals, ...

In Animals, the mRNA Export is facilitated by the TAP- p15 receptor

Vesicular Transport

Membrane enclosed transport intermediates ferry proteins from one compartment to the other

nuclear membrane

One of the membranes forming the nuclear envelope; the outer nuclear membrane is continuous with the endoplasmic reticulum and the inner nuclear membrane is adjacent to the nuclear lamina.

Ran-GTP binding results in...

Ran-GTP binding results in cargo release in nuclear import

Transmembrane Transport

Transmembrane protein translocators directly transport protein into a topologically distinct space

RNAs are transported...

to the cytoplasm as ribonucleoprotein complexes (RNPs).

Ran GTPase gives directionality to nuclear transport

•Molecular switch gives two conformational states ◦ Ran GTP ◦ Ran GDP

Ran Specific Regulatory Proteins Catalyze this transition

◦ Ran GEF ◦ Ran GAP

Ran GAP

◦ Ran GTPase Activating Protein (Ran GAP) : GTP-GDP

Ran GEF

◦ Ran Guanine Exchange Factor (Ran GEF) : Ran GDP-GTP

An electron micrograph of a nucleus

(A) An electron micrograph of a nucleus. The inner and outer nuclear membranes are joined at nuclear pore complexes (arrows).

An electron micrograph of the outer nuclear membrane with the endoplasmic reticulum

(B) An electron micrograph illustrating the continuity of the outer nuclear membrane with the endoplasmic reticulum.

Schematic of the nuclear envelope.

(C) Schematic of the nuclear envelope. The inner nuclear membrane is lined by the nuclear lamina, which serves as an attachment site for chromatin.

exportin

A karyopherin that recognizes nuclear export signals and directs transport from the nucleus to the cytosol.

importin

A karyopherin that recognizes nuclear localization signals and directs nuclear import.

nuclear pore complex

A large structure forming a transport channel through the nuclear envelope.

nuclear lamina

A meshwork of lamin filaments providing structural support to the nucleus.

karyopherin

A nuclear transport receptor.

nuclear transport receptor

A protein that recognizes nuclear localization signals and mediates transport across the nuclear envelope.

Ran

A small GTP-binding protein involved in nuclear import and export.

nuclear export signal

An amino acid sequence that targets proteins for transport from thenucleus to the cytosol.

nuclear localization signal

An amino acid sequence that targets proteins for transportation from the cytoplasm to the nucleus.

lamin

An intermediate fibrous filament protein that forms the nuclear lamina.

Distribution of Ran/GTP across the nuclear envelope

An unequal distribution of Ran/GTP across the nuclear envelope is maintained by the localization of Ran GTPase-activating protein (Ran GAP) in the cytoplasm and Ran guanine nucleotide exchange factor (Ran GEF) in the nucleus. In the cytoplasm, Ran GAP (bound to the cytoplasmic filaments of the nuclear pore complex) stimulates the hydrolysis of GTP bound to Ran, leading to the conversion of Ran/GTP to Ran/GDP. In the nucleus, Ran GEF (bound to chromatin) stimulates the exchange of GDP bound to Ran for GTP, leading to the conversion of Ran/GDP to Ran/GTP. Consequently, a high concentration of Ran/GTP is maintained within the nucleus.

Nuclear export

Complexes between cargo proteins bearing nuclear export signals (NES), exportins, and Ran/GTP form in the nucleus. Following transport through the nuclear pore complex, Ran GAP stimulates the hydrolysis of bound GTP, leading to formation of Ran/GDP and release of the cargo protein and exportin into the cytoplasm.

pore complexes have how many subunits?

Electron microscopy shows pore complexes have 8 subunits organized around a large central channel.

Multiple, low affinity associations

Multiple, low affinity associations between receptors and phenylalanine (F), glycine (G) repeats enable import receptors to navigate through the pore

Mutations in lamin

Mutations in lamin genes cause several different inherited tissue-specific diseases.

The nuclear pore complex Summary

Nuclear pore complexes are large structures that provide the only routes through which molecules can travel between the nucleus and the cytoplasm. Small molecules diffuse freely through the nuclear pore complex. Macromolecules are selectively transported either from nucleus to cytoplasm or from cytoplasm to nucleus.

Selective transport of proteins to and from the nucleus

Proteins destined for import to the nucleus contain nuclear localization signals that are recognized by receptors that direct transport through the nuclear pore complex. Proteins that shuttle back and forth between the nucleus and the cytoplasm contain nuclear export signals that target them for transport from the nucleus to the cytoplasm. In most cases, the small GTP-binding protein Ran is required for translocation through the nuclear pore complex and determines the directionality of transport. see ani 9.1

Gated Transport

Proteins move from cytosol to nucleus through the nuclearpore complex

Transport of RNAs

RNAs are transported through the nuclear pore complex as ribonucleoprotein complexes. Messenger RNAs, ribosomal RNAs, and transfer RNAs are exported from the nucleus to function in protein synthesis. Several classes of small nuclear RNAs are initially transported from the nucleus to the cytoplasm where they associate with proteins to form RNPs; they then return to the nucleus.

Random Mutageneis

Random, nonspecific amino acid changes

Molecular traffic through nuclear pore complexes

Small molecules are able to pass freely through the nuclear pore complex by passive diffusion. In contrast, macromolecules (proteins and RNAs) are recognized by specific signals and selectively transported from nucleus to cytoplasm or from cytoplasm to nucleus.

Site Directed Mutagenesis

Specific subsitution of one amino acid with another

Regulation of nuclear protein import

The activity of some proteins, such as transcription factors, is controlled by regulation of both their import to, and export from, the nucleus.

nuclear envelope

The barrier separating the nucleus from the cytoplasm, composed of an inner and outer membrane, a nuclear lamina, and nuclear pore complexes.

Model of lamin assembly

The lamin polypeptides form dimers in which the central a-helical regions of two polypeptide chains are wound around each other. Further assembly may involve the head-to-tail association of dimers to form linear polymers and the side-by-side association of polymers to form higher order structures.

Structure of the nuclear envelope

The nuclear envelope separates the contents of the nucleus from the cytoplasm, maintaining the nucleus as a distinct biochemical compartment that houses the genetic material and serves as the site of transcription and RNA processing in eukaryotic cells. The nuclear envelope consists of the inner and outer nuclear membranes (which are joined at nuclear pore complexes) and an underlying nuclear lamina.

Regulation of nuclear import of transcription factors

The transcription factor NF-kB is maintained as an inactive complex with IkB, which masks its nuclear localization sequence (NLS) in the cytoplasm. In response to appropriate extracellular signals, IkB is phosphorylated and degraded by proteolysis, allowing the import of NF-kB to the nucleus. The yeast transcription factor Pho4 is maintained in the cytoplasm by phosphorylation in the vicinity of its nuclear localization sequence. Regulated dephosphorylation exposes the NLS and allows Pho4 to be transported to the nucleus.

Protein import through the nuclear pore complex

Transport begins when the nuclear localization sequence (NLS) of a cargo protein is recognized by an importin. The importin/cargo complex binds to nuclear pore proteins in the cytoplasmic filaments and is transported through the pore. At the nuclear side of the envelope, Ran/GTP binds to the importin, disrupting the importin/cargo complex and releasing the cargo protein into the nucleus. The importin-Ran/GTP complex is re-exported through the nuclear pore and the GTPase-activating protein (Ran GAP) in the cytoplasm hydrolyzes the GTP on Ran to GDP, releasing the importin.

How are molecules imported into the nucleus?

• Nuclear Import Receptors are called Importins • Bind the NLS of cargo • Bind "FG" repeats along the path of the nuclear pore • Gradient of RAN Proteins drives movement across the membrane

RNAs are transported to the cytoplasm as ribonucleoprotein complexes (RNPs).

• RNAs are transported to the cytoplasm as ribonucleoprotein complexes (RNPs). • Karyopherin importins and exportins transport • tRNAs • rRNAs • miRNAs • small nuclear RNAs in a Ran/GTP-dependent manner. • Small nuclear RNA return to nucleus to function in mRNA processing

Single Amino Acid Subsitutions in the Laboratory

• Site Directed Mutagenesis • Random Mutageneis

Signal Sequences Direct Proteins to the Correct Address

• Typically 15-60 amino acid residues long • Can be found in the protein - N-terminus • Cleaved off by signal peptidases - Internal • Remain part of the protein • Multiple internal signals form 3D signal patch

Four facts about Lamins:

• bind to inner membrane proteins such as emerin and lamin B receptor (LBR). • are connected to the cytoskeleton by LINC complexes. • bind to chromatin. • extend in a loose meshwork throughout the interior of the nucleus.

mRNA are exported by a distinct mechanism

• does not involve karyopherins • is independent of RAN • Utilizes a distinct mRNA exporter complex of 20+ proteins • Helicase on the cytoplasm side releases the mRNA and ensures unidirectional transport

Ran-GTP binding results in cargo release in nuclear import

•At the nuclear side the cargo/importin complex is disrupted by binding of Ran/GTP. •This causes a conformational change in the importin, which releases the cargo protein into the nucleus. •partially covers conserved region important for cargo binding.

Model of the nuclear pore complex

•Eight spokes are connected to rings at the nuclear and cytoplasmic surfaces. •The spoke-ring assembly surrounds a central channel. •Protein filaments extend from the rings, forming a basket like structure on the nuclear side.

Proteins move between compartments three fundamentally different ways

•Gated Transport •Transmembrane Transport •Vesicular Transport

lamin dimers

•Mammals have 3 lamin genes (A, B, and C) which code for 7 proteins. •Two lamins interact to form a dimer: the α-helical regions wind around each other to form a coiled coil. •The lamin dimers associate with each other to form the lamina.

Size affects nuclear import

•NPC Proteins lining the pore have large, disordered structures: size filters •Small molecules (5,000 DA or less) diffuse freely •Proteins 5000 <60,000 Diffuse more slowly •Proteins > 60,000 Cannot pass without help, for ex: DNA Pol and RNA pol

Nuclear localization signals target proteins to the nucleus

•Nuclear import Signal: -aa-Lys-Lys-Lys-Arg-Lys-aa •Nuclear export signal: -Leu-aa-Leu-aa-aa-Leu-aa-aa-Leu-aa-Leu •Precise location does not seem to be important -N terminal or interior of primary sequence •NLS on one protein in a complex will result in translocation of all proteins

Uses of nuclear pore complex

•Nuclear pore complexes are composed of about 30 different pore proteins (nucleoporins). •RNAs synthesized in the nucleus must be exported to the cytoplasm for protein synthesis. •Proteins needed for nuclear functions must be imported from synthesis sites in the cytoplasm.

Protein export from the nucleus

•Proteins are targeted for export by amino acid sequences called nuclear export signals (NES). •NES are recognized by receptors in the nucleus (exportins), which direct protein transport to the cytoplasm. •Ran is also required for nuclear export. •Ran/GTP promotes binding of exportins and cargo proteins (but dissociates complexes between importins and cargos).

Gradient of two forms gives directionality to nuclear transport

◦ Higher concentration of Ran/GRP in the nucleus, determines directionality of transport