5. the nucleus: structure, nuclear transport Flashcards
Nucleus
storage and decoding of genetic information
Nucleus structure
- 10% of cell volume
- contains DNA- condensed and organized with proteins as chromatin
- Nuclear matrix- protein- containing fibrillar network
- nucleolus- rRNA synthesis, ribosome assembly
- surrounded by nuclear envelope
- Chromatin
Active Chromatin
euchromatin appear lighter
inactive chromatin
heterochromatin is dark and located near periphery of nucleus
nucleolus
a granular region
site of ribosome assembly
nuclear envelope: 2 lipid bilayer membranes, 10-50 nm apart
- the outer membrane is continuous with and endoplasmic reticulum
- a denser layer of intermediate filaments, the nuclear lamina, is within the inner membrane
nuclear lamina
- composed of lamin proteins
- provides structure to nucleus, heterochromatin binding site
- Defect in lamin A gene results in progeria-premature again
normal nucleus lamin A distribution
perfectly oval
progeria nucleus lamin A distribution
rigid circle
Nuclear Pores ~3000/nucleus
- how the cell moves molecules into and out of the nucleus
- at the pore, the inner and outer membranes come together forming an opening
- opening is lined with proteins: nuclear pore complex
nuclear pore complex
- at least 30 different nucleoporin proteins, in octagonal; symmetry
- including cytoplasmic filaments, extending to the cytoplasm
The role of nuclear pores
-regulate transport of RNA and proteins into and out of nucleus
act as turnstiles
-traffic is FAST! 1 typical human cell has to import 560,00 ribosomal proteins/minute, export 14,000 assembled ribosomes/minute
-small molecules and proteins of less than 9nm can pass freely
-large proteins must be assisted
Energy-dependent transport:
RNAs
most proteins
Passive diffusion:
ions
small molecules
water
How is the import or export of large proteins regulated?
nuclear pores regulate traffic into and out of the nucleus by means of the nuclear localization signal (NLS)- (the ticket for the turnstile)
- a specific amino acid sequence marks protein for nuclear entry
- a series of +ively charged amino acids in specific sequence:
- pro-lys-lys-lys-arg-lys-val-
NLS is in the ________
protein
How do we know the function of the NLS?
experiment were amino acid sequence is modified (by site-directed-mutagenesis)
- “SV40 virus T-antigen” a viral protein
- normally enter the nucleus if injected into cytoplasm (because it has adopted an NLS-counterfeit ticket?)
- this protein normally accumulates in the nucleus
- experimentally visualized the location of the protein using a fluorescent tag which glows under UV light
- set up a microscope with ultraviolet light source
- an experimental alteration of the sequence: (pro-lys-THR-lys-arg-lys-val-) completely prevents accumulation in the nucleus
What happens when we alter the NLS?
in 1984, kalderson and colleagues mutated a single amino acid of a protein’s NLS
- wild type- green labeled protein is in the nucleus and not cytoplasm
- green labeled protein is in the cytoplasm and not in the nucleus
What happens when we use recombinant DNA techniques to add the NLS to a dummy protein??
normal or modified bovine serum albumin (NLS added) and injected to the cytoplasm
- This provided evidence of nuclear transport receptors
- family of proteins associated with the nuclear pore complex
- importins recognize the NLS and bring proteins in
- another set of proteins, the exportins, work in the opposite direction
- these recognize other signals
Diffusion
- while some movement through the nuclear pore is regulated by a gating system, small molecules enter and exit by diffusion
- diffusion-movement of molecules from a regions of high concentration to a low concentration (down a concentration gradient)
- note that each molecule will typically follow its own concentration gradient
Mechanism of protein import through nuclear pore complex has how many steps ?
8
protein import through nuclear pore step 1
protein binds to a two-protein complex (importin alpha and importin beta)
importin alpha is a receptor for the NLS portion of the protein to be imported
ie the NLS recognizes and sticks to importin alpha
protein import through nuclear pore step 2
importin complex and protein stick to cytoplasmic filament
-mediated by importin beta
protein import through nuclear pore step 3
complex moves into nucleoplasm
-not an energy consumer step, it can go back at this point unless captured by the RanGTP in next step.
protein import through nuclear pore step 4
complex bind to another protein
- this is the RanGTP; after binding RanGTP, complex dissociates
- importin beta stays on the RanGTP
protein import through nuclear pore step 5
Ran-GTP-importin beta complex moves back to the cytoplasm, down a concentration gradient
protein import through nuclear pore step 6
now two things happen
- the Ran GTP is converted to Ran GDP plus phosphate by the enzyme RanGAP. this causes RanGTP to release importin Beta
- an exportin molecule binds to exportin molecule binds to importin alpha, setting it up for transport out of the nucleus
protein import through nuclear pore step 7
RanGDP diffuses back to the nucleus down ITS concentration gradient, from high to low concentration
-exportin carries importin alpha out of the nucleus
protein import through nuclear pore step 8
restoration to initial state
- the importin alpha and importin beta complex re-forms
- enzyme RCC1 reforms RanGDP to RanGTP
GDP->GTP is energy ______
consuming
GTP-> GDP is energy _______
producing
GTP to GDP conversion controls the process
- molecules always diffuse from high to low concentration, so a steep gradient of RanGTP across the nuclear membrane drives the process
- 2 enzymes on either side of membrane are responsible for maintaining gradient: RCC1 occurs only in the nucleoplasm, RanGAP in cytoplasm
RanGAP maintains low conc. of Ran-GTP in the _________.
cytoplasm
RCC1 maintains the high con. of Ran GTP in the _______
nucleus
Ran-GTP continues to diffuse from the ______
nucleus
interior of nuceleus
DNA
DNA replications
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