5. the nucleus: structure, nuclear transport Flashcards Preview

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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


Active Chromatin

euchromatin appear lighter


inactive chromatin

heterochromatin is dark and located near periphery of nucleus



a granular region
site of ribosome assembly


nuclear envelope: 2 lipid bilayer membranes, 10-50 nm apart

1. the outer membrane is continuous with and endoplasmic reticulum
2. 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:

most proteins


Passive diffusion:

small molecules


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:


NLS is in the ________



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



-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 ?



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 ______