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Cell Biology > 5. the nucleus: structure, nuclear transport > Flashcards

5. the nucleus: structure, nuclear transport Flashcards

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1
Q

Nucleus

A

storage and decoding of genetic information

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2
Q

Nucleus structure

A
  • 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
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3
Q

Active Chromatin

A

euchromatin appear lighter

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4
Q

inactive chromatin

A

heterochromatin is dark and located near periphery of nucleus

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5
Q

nucleolus

A

a granular region

site of ribosome assembly

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6
Q

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

A
  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
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7
Q

nuclear lamina

A
  • composed of lamin proteins
  • provides structure to nucleus, heterochromatin binding site
  • Defect in lamin A gene results in progeria-premature again
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8
Q

normal nucleus lamin A distribution

A

perfectly oval

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9
Q

progeria nucleus lamin A distribution

A

rigid circle

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10
Q

Nuclear Pores ~3000/nucleus

A
  • 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
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11
Q

nuclear pore complex

A
  • at least 30 different nucleoporin proteins, in octagonal; symmetry
  • including cytoplasmic filaments, extending to the cytoplasm
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12
Q

The role of nuclear pores

A

-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

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13
Q

Energy-dependent transport:

A

RNAs

most proteins

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14
Q

Passive diffusion:

A

ions
small molecules
water

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15
Q

How is the import or export of large proteins regulated?

A

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-
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16
Q

NLS is in the ________

A

protein

17
Q

How do we know the function of the NLS?

A

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
18
Q

What happens when we alter the NLS?

A

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
19
Q

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

A
  • 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
20
Q

Diffusion

A
  • 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
21
Q

Mechanism of protein import through nuclear pore complex has how many steps ?

A

8

22
Q

protein import through nuclear pore step 1

A

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

23
Q

protein import through nuclear pore step 2

A

importin complex and protein stick to cytoplasmic filament

-mediated by importin beta

24
Q

protein import through nuclear pore step 3

A

complex moves into nucleoplasm

-not an energy consumer step, it can go back at this point unless captured by the RanGTP in next step.

25
Q

protein import through nuclear pore step 4

A

complex bind to another protein

  • this is the RanGTP; after binding RanGTP, complex dissociates
  • importin beta stays on the RanGTP
26
Q

protein import through nuclear pore step 5

A

Ran-GTP-importin beta complex moves back to the cytoplasm, down a concentration gradient

27
Q

protein import through nuclear pore step 6

A

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
28
Q

protein import through nuclear pore step 7

A

RanGDP diffuses back to the nucleus down ITS concentration gradient, from high to low concentration
-exportin carries importin alpha out of the nucleus

29
Q

protein import through nuclear pore step 8

A

restoration to initial state

  • the importin alpha and importin beta complex re-forms
  • enzyme RCC1 reforms RanGDP to RanGTP
30
Q

GDP->GTP is energy ______

A

consuming

31
Q

GTP-> GDP is energy _______

A

producing

32
Q

GTP to GDP conversion controls the process

A
  • 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
33
Q

RanGAP maintains low conc. of Ran-GTP in the _________.

A

cytoplasm

34
Q

RCC1 maintains the high con. of Ran GTP in the _______

A

nucleus

35
Q

Ran-GTP continues to diffuse from the ______

A

nucleus

36
Q

interior of nuceleus

A

DNA
DNA replications
Transcription

Cell Biology (16 decks)

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