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HELENP CBG: Cell Architecture, Communication and Regulation > CBG Lecture 33&34: Cell Architecture and Nuclear Transport > Flashcards

CBG Lecture 33&34: Cell Architecture and Nuclear Transport Flashcards

1
Q

what are main organelles of eukaryotic cell

A 
nucleus
SER/RER
Golgi apparatus (&golgins)
Peroxisomes
Lysosomes
Endosomes
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2
Q

outline the structure of the nucleus

A

nucleolus, nucleoplasm,nuclear lamina and innteer/outer nuclear envelope, perinuclear space with pores

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3
Q

what is the nucleae lamina

A

gives nucleus structure- filamentous proteins that form a meshwork on the inside of the inner nuc,ear membrane providing anchorage sites for chroms and nuclear pore

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4
Q

what happens to the nuclear lamina during mitosis

A

disassmebled and interacts with chromatin

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5
Q

what holds chromosomes in a non random arrangement

A

interaction of chroms with lamins holding chroms in fixed position in nucleoplasm

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6
Q

what controls the transort in and out of the nucleus

A

nuclear pore complex

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7
Q

what is the nucleus directly connected to

A

RER

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8
Q

what does concentration of NPCs of a nucleus depend on

A

increase nucleus activity, increase concentration NPCs

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9
Q

how many NPCs does a typical nucleus contain

A

3k-4k

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10
Q

what is role of the nucleus

A

half the cell mass
store DNA : euks-nucleus and endosymbionts, proks:nucleoid and plasmid
protection of genome from dynamic cytoskeleton and protection from condensation of genome during mitosis
regulate gene expression - TFs localise to nucleus
processing mRNA
bidirectional traffic
create ribosomes, mRNA and tRNA (nucleolus) ribosome constuction = rRNA and proteins - protein manufacturing in cytosol

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11
Q

how does nucleus protect the genome

A

from dynamic skeleton - causes condensation during mitosis

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12
Q

discuss the atomic-scale structure of the ribosome

A

LSU - 49proteins &2RNA

SSU = 33 proteins&1RNA molecule

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13
Q

what is the nuclear lamina

A

meshwork of interocnnected proteins called lamins that give shape and stability
interacts with proteins on inner nuclesar membrane
interacts with chromatin
disassembled during mitosis

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14
Q

what is an NPC

A

nuclear pore complex

v big macromolecule 125MDa - composed of 30 different nuclearporin proteins

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15
Q

what is structure of NPC

A

3 main parts

  1. cytoplasmic fibrils
  2. central framework
  3. nuclear basket
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16
Q

what is inside the central framework of the NPC

A

an FG (phenol&glycine rich) meshwork core: transport conduit - permeability barrier

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17
Q

what was used to determine architecture of NPC

A

crosslinking of neighbouring proteins)
mass spec/proteomics
Immuno EM
analytical ultracentrifugation

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18
Q

how to separate RER from SER

A

by centrifugation

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19
Q

what is the ER

A

a network of interconnected branching tubules extending throughout the cytosol

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20
Q

how much of total cell volume does ER cisternal space occupy

A

Er lumen occupies&raquo_space;10%

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21
Q

what is ERs major contribution to cell

A

Er membrane makes major contribution to mitochondrial and peroxisomal membranes by producing most of their lipids

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22
Q

where are almost all of the proteins that will be secreted to the cell exterior and those destined for lumen of Golgi/lyossomes initially secreted to

A

ER - ER captires selected proteins from the cytosol as they’re being syntheszed

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23
Q

what makes RER different from SER

A

has membrane bound ribosomes that coat the surface

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24
Q

what type of ER do hepatocytes have

A

SER

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25
Q

what are SERs involved in

A

lipid and carb metabolism

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26
Q

what do SERs sequester

A

Ca2+ from cytosol - important for muscle cell contraction

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27
Q

what is a Golgi Apparatus

A

organised ordered stacks of disklike compartments called cisternae which receive lipid proteins from ER and dispatches them

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28
Q

why does Golgi have directionality

A

cis-trans faces for vesicle transportation

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29
Q

what does the structural integrity of Golgi depend on

A

microtubules and cytoskeleton

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30
Q

what helps organise the Golgi stack of cisternae

A

the Golgi matrix

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31
Q

what is function of Golgi

A

carb synthesis
sorting and dispatching cargo from ER
microtubule nucleation

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32
Q

what are golgins

A

peripheral membrane proteins associated with cytoplasmic side of Golgi membranes

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33
Q

what are the peripheral membrane proteins associated with cytoplasmic side of Golgi membranes called

A

Golgins

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34
Q

what structure do golgins have

A

coiled coil proteins which form long filaments up to 200nm with diverse binding partners

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35
Q

how long can golgins get

A

up to 200nm

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36
Q

what are peroxisomes

A

spherical and vesicular organelle which contains enzymes and is enclosed by a lipid bilayer - roteins for waste disposal get suhffled here

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37
Q

what reactions are peroxisomes important for

A

oxidative reactions - energy metabolism - breakdown long chain fatty acids

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38
Q

what are lysosomes

A

spherical vesicles enclosed by a lipid bilayer with ph

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39
Q

what are endosomes

A

a type is a lyssome -sorting place like Golgi
spherical, vary in size
enclosed by lipid bilayer

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40
Q

what types of endosomes are there

A

early - sorting
recycling - return to PM
late - target for degradation

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41
Q

what pathway do internalised molecules from PM follow to get to lysosomes for degradation

A

the endocytic membrane transport pathway

42
Q

what happens to proteins with no signal sequence

A

cytoplasmic

43
Q

what happens to proteins with NLS

A

nuclear localization sequence - nuclear

44
Q

what happens to proteins with ER signal sequence

A 
plasma membrane
secreted
resident ER and Golgi
endosomes
lysosomes
45
Q

what do sorting signals doi

A

directs protein delivery to locations outside the cytosol or to organelle surfaces

46
Q

how long are typical sorting signals

A

15-60 residues long found at the N terminus of the pp chain`

47
Q

what do signal peptidases do

A

remove the signal sequence ffrom the finished protein once the sorting process is complete

48
Q

which signals are used in gated transport into the nucleus? what are the stretches of aas like

A

signal sequences with internal stretches of amino acids which remain part of the protein are used in gated transport into the nucleus

49
Q

what does a typical signal sequence for proteins destined for the ER contain

A

usually have a sequence of about 5-10 hydrophobic aas on their N terminus

50
Q

what does a typical sorting signal for proteins destined for the mitochondria contain

A

alternating positively charged with hydrophobic aas

51
Q

what is nucleus signal sequence typically made of

A

PPKKKAKV

52
Q

what is the volume of vtraffic of NPCs - direction? receptors?

A

1000macromolecules/sec
bidirectional transport
passive diffusion for small things
specific receptors (karyopherins) ferry large protesins (AT): importins (alpha and beta subunits), exportins, FG-Nups

53
Q

what are specific NPC receptos called

A

karyopherins

54
Q

what are karyopherins

A

specific NPC receptors

55
Q

how was the mechanism for nuclear transport determined (AT)

A

took homopentamer nucleoplasmin (each monomer has tail) and injected it into Xenopus (frog)oocytes - w/wo proteolysis
also injected into cytosol - nucleus
the number of tails on pentamer correlates with rate of transport
if injected into nucleus, it stays there
tail alone goes into nucleus therefore tail likely contains NLS

56
Q

what is nucleoplasmin

A

the most abundant protein of the nucleus of Xenopus laevis oocytes

57
Q

what is the most abundant protein of Xenopus oocyte nucleus

A

nucleoplasmin

58
Q

how was nucleoplasmin used to determine the mechanism for nucleotransport (AT)

A

injected homopentamer nucleoplasmin into nucleus, and tail stayed there - likely to contain NLS

59
Q

what must most NLSs be recognised by to initiate nuclear import

A

IMPORTINS - nuclear import erceptors

60
Q

what are importins

A

soluble cytosolic proteins that bind oth to the NLS on the cargo protein adn the FG phenylalanine glycine repeats in the unstructured domains of the channel nucleoporins that line the central pore

61
Q

what gives FG repeats gel like properties

A

in the unstructured tangle of the pore they interact weakly giving gel like properties that impose a permeability barrier to large macromolecules - also serve as docking sites for importins

62
Q

how was the receptors for import signals discovered

A

treat oocytes with digitonin (detergent) which permeabilises PM, keeping nuclear envelopes in tact but nuclei retained the ability to transport and accumulate proteins
transport required addition of cytosol to permeabilized cells, indicating the soluble cytoplasmic factors required for nuclear import are released during digitonin treatment
Nuclear import is specific for a functional NLS and requires ATP and cytosol and is temperature dependent

63
Q

what was digitonin used for - what did it do

A

used to discover receptors for import signals - permeabilizes the OM, keeping nuclear envelopes intact

64
Q

what does nuclear import require

A

ATP
cytosol
functional NLS
temperature dependent

65
Q

how is accumulation of the cargo within the nucleus inhibited

A

by wheat germ agglutinin which binds NPCs and inhibits trnsport

66
Q

what does wheat germ agglutinin do

A

binds to NPCs and inhibits cargo transport

67
Q

name some small G proteins

A

Ran
GEF
GAP

68
Q

what imposes directionality on transport through NPCs

A

Ran GTPase

69
Q

how does the cell fuel the import of nuclear proteins through NPCs

A

by harnessing energy stored in concentration gradients of GTP bounds form of the monomeric GTPase Ran, which is required for both nuclear import and export

70
Q

what is Ran

A

a GTPase molecular switch that can coexist in two conformational states, depending on whether GDP or GTP is bound

71
Q

what Ran secific regulatory proteins trigger the conversion between GDP/GTP bound Ran

A

a cytosolic GAP (GTPase Activating Protein)

a nucear GEF -guanine exchange factor

72
Q

where is GEF

A

guanine exchange factor found in nucleus

73
Q

where is GAP

A

GTPase Activating protein found in cytosol

74
Q

what does GAP do

A

GTP activating protein triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP

75
Q

what does GEF do

A

converts Ran-GDP to Ran-GTP

76
Q

what contains more Ran-GTP, nucleus or cytosol - why?

A

nucleus contains mainly ran-GTP because Ran-GEF is located in nucleus where it is anchored to chromatin

77
Q

what is nuclear transport driven by

A

GTP hydrolysis

78
Q

how is nuclear transport driven by GTP hydrolysis

A
  1. cargo binds to importin in cytosol and receptor-cargo complex goes through nuclear pore into nucleoplasm
  2. only large proteins with NLS can enter nucleu
  3. NLS normally PPKKKRKV
  4. NLS recognised by importins, which carry cargo into nucleus
  5. with cargo attached to alpha importin, importin binds to specific nuclear pore proteins in the cytoplasmic filaments
  6. by sequential binding to more interior NPPs the complex is translocated through the nuclear pore
  7. at the nuclear side of the pore, the complex is disrupted by the binding of Ran to importin
  8. Ran carries GTP, binding changes conformation of importin which then releases its cargo protein into the nucleus
  9. Importin-Ran complex is then re-exported through the nuclear pore
  10. Ran-GAP stimulates Ran to hydrolyse its GTP to GDP, triggering Ran to release importin back into the cytoplasm
79
Q

what are the subunits of importins

A

alpha

beta

80
Q

describe alpha importin

A

series of repeat units

armadillo motif which binds to NLS, crystal structure allows importin recognition of NLS tails

81
Q

descibe beta importin

A

armadillo motif
binds to Fg repeats that are in Fg meshwork
has scaffold on opposite face to FG repeat, where RAN-GTP binds

82
Q

what models for cargo transport through NPC exist

A
  1. Selective phase-hydrogel model
  2. virtual gate/polymer brush model
  3. forest model
  4. reduction of dimensionality model
83
Q

what are FG repeats

A

abundand phenylalanine glycine rich repeat domains that form spaghetti like meshwork within NPCs - they represent binding sites for nuclear transport receptors

84
Q

what happens to lamins during prometaphase

A

theyre phosphorylated

85
Q

name some molecules that need to get out of nucleus

A

mRNA ribosomes

86
Q

name some molecules that need to get into nucleus

A 
DNAP
RNAP 
histones
lamins
steroids
87
Q

what fold symmetry do nuclear pores have whenviewed from above

A

8-fold symmetry

88
Q

discuss selective phase/hydrogel model of protein trafficking through nucleus

A

diffusion barrier of the NPC is a 3D seive which allows the translocation of small molecules but restricts larger ones
Nuclear Transport receptor partitions into meshowrk buy weak interactions
mesh size determined by hydrophobic clusters within Fg-Nups which form the hydrogen - also by concn of Fg-Nups and distance between individial
permeability barrier can be reproduced in vitro

89
Q

in selective phase model, how does NTR partition into meshwork

A

by weak interactions

90
Q

what polarity are FG clusters in the selective phase model

A

NTR bind the hydrophobic clusters of the FG-rich repeats and hence compete with inter repeat contacts

91
Q

discuss the Forest model - what is it based on

A

based on observed hydrodynamic radius of FG-Nups
collapsed coils - shrubs
extended coils 0 trees
trees have collapsed coil domain separated by extended coil

92
Q

discuss the virtual gate model/polymer brush model

A

based on thermodynamic considerations
- as cargo passes through the constriction of the channel it loses entropy, FG filaments act as brush bristles move back and forth to hinder the transport
makes an entropic gate
if the binding of molecules with FG-regions of nucleoporins is high enough, it might compensate for the loss in entropy
NPCs lower the activation energy thats required to allow entry of a molecule into the narrow pore
based on atomic force microstudies of surface tethered FG-Nups

93
Q

which nuclear protein trafficking model does not depend on FG-FG interactions

A

the polymer brush/virtual gate model

94
Q

discuss reduction of dimensionality model

A

assume FG-Nups are saturated with NTRs - evidence is unclear
FG-Nups are in a permanent state of collapse
passive diffusion of small molecules through the central channel
cargo goes through random 2D walk along wlalls of channel displacing cargo free NTRs

95
Q

what is the reduction of dimensionality model based on

A

flourescent correlation spectroscopy study showing FG-Nups have a high number of NTRs. some immobile

96
Q

what are most likely cargo trafficking models

A

selective phase

virtual gate

97
Q

what is the RER NLS return

A

KDEL

98
Q

what is the mitochondrion signalling sequence

A

Amphipathic helix

99
Q

which organelle does the signal peptide: Positive-hydrophobic-negative/polar direct to

A

RER

100
Q

what do all the components of Positive-hydrophobic-negative/polar function for

A

positive is the NH3+ terminus of the nascent polypeptide, hydrophobic anchors the protein to the RER membrane and polar disengages it

HELENP CBG: Cell Architecture, Communication and Regulation (5 decks)

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