CMB Exam 4

Nucleus

What does the nuclear envelop consist of

The inner and outer membranes that are joined at nuclear pore complexes

The outer membrane of the nuclear envelope is contiguous with the endoplasmic reticulum

What is the nuclear lamina

A meshwork of intermediate filaments that provides structural support for the nucleus

What is the model of lamin assembly

Lamin polypeptides coil to form a dimer.

Head to tail association of dimers combine to form a dimer

Side by side association of polymers will form the higher order structure of the nuclear membrane

Prenylation

targets Lamins to inner nuclear membrane

Hutchinson-Gilford Progeria Syndrome

Dominant negative effect of mutant lamin A on lamina function. Essentially nucleus is wonky and kid ends up with a bunch of problems and a big head

How do small molecules pass through the NPC

Small molecules go through passive diffusion

How do macromolecules such as proteins and RNAs transport through the NPC

It is a selective, energy-dependent process

Central channel of the NPCs

Occupied by FG-repeat nups

~200 FG-nups/NPC form a flexible, highly amorphous meshwork

FG-nups serve as binding sites for nuclear transport receptors (importins and exportins)

About the NPCs??

Vertebrate cells typically contain 1000-10000 NPCs/cell

Molecular weight cutoff ~30-40 kD

NPC contains ~30 different protein subunits (nucleoporins or nups)

Protein import through the NPC

1. Cargo contains a nuclear localization sequence (NLS) that acts like a zip code for protein import

2. Importin binds to the NLS and brings it through the NPC into the nucleus, where cargo is then released

3. Ran-GTP binds to importin and propels through the NPC into the cytoplasm using a GTPase activating protein Ran-GAP. The importin is then released

4. Ran-GDP is recycled back to the nucleus using NTF2 (importin protein).

5. Once Ran-GDP is back in the nucleus, it is phosphorylated into Ran-GTP with a Ran GEF (guanine nucleotide exchange factor) where it can be active n shit

Cytoplasm - low Ran/GTP

Nucleus - high Ran/GTP

Nuclear Localization signal (NLS)

Comprised of single or bipartile segments of basic amino acids

It is recognized by importin proteins (nuclear transport receptors)

Export through the nuclear pore complex (NPC)

1. Ran-GTP, NES (nuclear export signal), and cargo protein form a threesome.

2. Triplet is propelled through the NPC into the cytoplasm using the Ran GAP, which will dissociate them

3. Cargo is released, Ran-GDP binds to NTF2, that complex binds to exportin

4. Threesome travels through the NPC using Ran GAP. Dissociates once in nucleus.

5. Ran-GDP is phosphorylated back into Ran-GTP by Ran GEF

Transport of snRNAs between the nucleus and cytoplasm

1. snRNA-Crm1 bound in nucleus, travels through NPC

2. Complex dissociates in cytoplasm

3. snRNA binds to proteins → snRNP

4. Snurportin binds to snRNP and transports through NPC into nucleus.

5. Snurportin dissociates

Transcription factor import in response to cell signaling

1. NF-kB/NLS/IkB is phosphorylated and proteolyzed (IkB dissociates), allowing importin to attach

2. Proceeds through NPC into nucleus

or

1. Pho4/NLS/P_i is dephosphorylated (unmasks NLS), allowing importin to attach

2. Proceeds through NPC into nucleus

Induction of androgen receptor nuclear import by androgen

Signal = androgen diffuses across plasma membrane into cytoplasm

Androgen binds to an androgen receptor. This complex dissociates and dimerizes, allowing nuclear transport

Dimer binds to DNA and activates transcription

Distribution of transcriptionally active and inactive chromatin

NAD - nucleolus associated domains

LAD - lamina associated domains

Transcription factories

Circle of transcriptionally-active chromatin through RNA polymerase and transcription factors