CBG Lecture 33&34: Cell Architecture and Nuclear Transport Flashcards
what are main organelles of eukaryotic cell
nucleus SER/RER Golgi apparatus (&golgins) Peroxisomes Lysosomes Endosomes
outline the structure of the nucleus
nucleolus, nucleoplasm,nuclear lamina and innteer/outer nuclear envelope, perinuclear space with pores
what is the nucleae lamina
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
what happens to the nuclear lamina during mitosis
disassmebled and interacts with chromatin
what holds chromosomes in a non random arrangement
interaction of chroms with lamins holding chroms in fixed position in nucleoplasm
what controls the transort in and out of the nucleus
nuclear pore complex
what is the nucleus directly connected to
RER
what does concentration of NPCs of a nucleus depend on
increase nucleus activity, increase concentration NPCs
how many NPCs does a typical nucleus contain
3k-4k
what is role of the nucleus
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
how does nucleus protect the genome
from dynamic skeleton - causes condensation during mitosis
discuss the atomic-scale structure of the ribosome
LSU - 49proteins &2RNA
SSU = 33 proteins&1RNA molecule
what is the nuclear lamina
meshwork of interocnnected proteins called lamins that give shape and stability
interacts with proteins on inner nuclesar membrane
interacts with chromatin
disassembled during mitosis
what is an NPC
nuclear pore complex
v big macromolecule 125MDa - composed of 30 different nuclearporin proteins
what is structure of NPC
3 main parts
- cytoplasmic fibrils
- central framework
- nuclear basket
what is inside the central framework of the NPC
an FG (phenol&glycine rich) meshwork core: transport conduit - permeability barrier
what was used to determine architecture of NPC
crosslinking of neighbouring proteins)
mass spec/proteomics
Immuno EM
analytical ultracentrifugation
how to separate RER from SER
by centrifugation
what is the ER
a network of interconnected branching tubules extending throughout the cytosol
how much of total cell volume does ER cisternal space occupy
Er lumen occupies»_space;10%
what is ERs major contribution to cell
Er membrane makes major contribution to mitochondrial and peroxisomal membranes by producing most of their lipids
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
ER - ER captires selected proteins from the cytosol as they’re being syntheszed
what makes RER different from SER
has membrane bound ribosomes that coat the surface
what type of ER do hepatocytes have
SER
what are SERs involved in
lipid and carb metabolism
what do SERs sequester
Ca2+ from cytosol - important for muscle cell contraction
what is a Golgi Apparatus
organised ordered stacks of disklike compartments called cisternae which receive lipid proteins from ER and dispatches them
why does Golgi have directionality
cis-trans faces for vesicle transportation
what does the structural integrity of Golgi depend on
microtubules and cytoskeleton
what helps organise the Golgi stack of cisternae
the Golgi matrix
what is function of Golgi
carb synthesis
sorting and dispatching cargo from ER
microtubule nucleation
what are golgins
peripheral membrane proteins associated with cytoplasmic side of Golgi membranes
what are the peripheral membrane proteins associated with cytoplasmic side of Golgi membranes called
Golgins
what structure do golgins have
coiled coil proteins which form long filaments up to 200nm with diverse binding partners
how long can golgins get
up to 200nm
what are peroxisomes
spherical and vesicular organelle which contains enzymes and is enclosed by a lipid bilayer - roteins for waste disposal get suhffled here
what reactions are peroxisomes important for
oxidative reactions - energy metabolism - breakdown long chain fatty acids
what are lysosomes
spherical vesicles enclosed by a lipid bilayer with ph
what are endosomes
a type is a lyssome -sorting place like Golgi
spherical, vary in size
enclosed by lipid bilayer
what types of endosomes are there
early - sorting
recycling - return to PM
late - target for degradation
what pathway do internalised molecules from PM follow to get to lysosomes for degradation
the endocytic membrane transport pathway
what happens to proteins with no signal sequence
cytoplasmic
what happens to proteins with NLS
nuclear localization sequence - nuclear
what happens to proteins with ER signal sequence
plasma membrane secreted resident ER and Golgi endosomes lysosomes
what do sorting signals doi
directs protein delivery to locations outside the cytosol or to organelle surfaces
how long are typical sorting signals
15-60 residues long found at the N terminus of the pp chain`
what do signal peptidases do
remove the signal sequence ffrom the finished protein once the sorting process is complete
which signals are used in gated transport into the nucleus? what are the stretches of aas like
signal sequences with internal stretches of amino acids which remain part of the protein are used in gated transport into the nucleus
what does a typical signal sequence for proteins destined for the ER contain
usually have a sequence of about 5-10 hydrophobic aas on their N terminus
what does a typical sorting signal for proteins destined for the mitochondria contain
alternating positively charged with hydrophobic aas
what is nucleus signal sequence typically made of
PPKKKAKV
what is the volume of vtraffic of NPCs - direction? receptors?
1000macromolecules/sec
bidirectional transport
passive diffusion for small things
specific receptors (karyopherins) ferry large protesins (AT): importins (alpha and beta subunits), exportins, FG-Nups
what are specific NPC receptos called
karyopherins
what are karyopherins
specific NPC receptors
how was the mechanism for nuclear transport determined (AT)
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
what is nucleoplasmin
the most abundant protein of the nucleus of Xenopus laevis oocytes
what is the most abundant protein of Xenopus oocyte nucleus
nucleoplasmin
how was nucleoplasmin used to determine the mechanism for nucleotransport (AT)
injected homopentamer nucleoplasmin into nucleus, and tail stayed there - likely to contain NLS
what must most NLSs be recognised by to initiate nuclear import
IMPORTINS - nuclear import erceptors
what are importins
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
what gives FG repeats gel like properties
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
how was the receptors for import signals discovered
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
what was digitonin used for - what did it do
used to discover receptors for import signals - permeabilizes the OM, keeping nuclear envelopes intact
what does nuclear import require
ATP
cytosol
functional NLS
temperature dependent
how is accumulation of the cargo within the nucleus inhibited
by wheat germ agglutinin which binds NPCs and inhibits trnsport
what does wheat germ agglutinin do
binds to NPCs and inhibits cargo transport
name some small G proteins
Ran
GEF
GAP
what imposes directionality on transport through NPCs
Ran GTPase
how does the cell fuel the import of nuclear proteins through NPCs
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
what is Ran
a GTPase molecular switch that can coexist in two conformational states, depending on whether GDP or GTP is bound
what Ran secific regulatory proteins trigger the conversion between GDP/GTP bound Ran
a cytosolic GAP (GTPase Activating Protein)
a nucear GEF -guanine exchange factor
where is GEF
guanine exchange factor found in nucleus
where is GAP
GTPase Activating protein found in cytosol
what does GAP do
GTP activating protein triggers GTP hydrolysis and thus converts Ran-GTP to Ran-GDP
what does GEF do
converts Ran-GDP to Ran-GTP
what contains more Ran-GTP, nucleus or cytosol - why?
nucleus contains mainly ran-GTP because Ran-GEF is located in nucleus where it is anchored to chromatin
what is nuclear transport driven by
GTP hydrolysis
how is nuclear transport driven by GTP hydrolysis
- cargo binds to importin in cytosol and receptor-cargo complex goes through nuclear pore into nucleoplasm
- only large proteins with NLS can enter nucleu
- NLS normally PPKKKRKV
- NLS recognised by importins, which carry cargo into nucleus
- with cargo attached to alpha importin, importin binds to specific nuclear pore proteins in the cytoplasmic filaments
- by sequential binding to more interior NPPs the complex is translocated through the nuclear pore
- at the nuclear side of the pore, the complex is disrupted by the binding of Ran to importin
- Ran carries GTP, binding changes conformation of importin which then releases its cargo protein into the nucleus
- Importin-Ran complex is then re-exported through the nuclear pore
- Ran-GAP stimulates Ran to hydrolyse its GTP to GDP, triggering Ran to release importin back into the cytoplasm
what are the subunits of importins
alpha
beta
describe alpha importin
series of repeat units
armadillo motif which binds to NLS, crystal structure allows importin recognition of NLS tails
descibe beta importin
armadillo motif
binds to Fg repeats that are in Fg meshwork
has scaffold on opposite face to FG repeat, where RAN-GTP binds
what models for cargo transport through NPC exist
- Selective phase-hydrogel model
- virtual gate/polymer brush model
- forest model
- reduction of dimensionality model
what are FG repeats
abundand phenylalanine glycine rich repeat domains that form spaghetti like meshwork within NPCs - they represent binding sites for nuclear transport receptors
what happens to lamins during prometaphase
theyre phosphorylated
name some molecules that need to get out of nucleus
mRNA ribosomes
name some molecules that need to get into nucleus
DNAP RNAP histones lamins steroids
what fold symmetry do nuclear pores have whenviewed from above
8-fold symmetry
discuss selective phase/hydrogel model of protein trafficking through nucleus
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
in selective phase model, how does NTR partition into meshwork
by weak interactions
what polarity are FG clusters in the selective phase model
NTR bind the hydrophobic clusters of the FG-rich repeats and hence compete with inter repeat contacts
discuss the Forest model - what is it based on
based on observed hydrodynamic radius of FG-Nups
collapsed coils - shrubs
extended coils 0 trees
trees have collapsed coil domain separated by extended coil
discuss the virtual gate model/polymer brush model
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
which nuclear protein trafficking model does not depend on FG-FG interactions
the polymer brush/virtual gate model
discuss reduction of dimensionality model
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
what is the reduction of dimensionality model based on
flourescent correlation spectroscopy study showing FG-Nups have a high number of NTRs. some immobile
what are most likely cargo trafficking models
selective phase
virtual gate
what is the RER NLS return
KDEL
what is the mitochondrion signalling sequence
Amphipathic helix
which organelle does the signal peptide: Positive-hydrophobic-negative/polar direct to
RER
what do all the components of Positive-hydrophobic-negative/polar function for
positive is the NH3+ terminus of the nascent polypeptide, hydrophobic anchors the protein to the RER membrane and polar disengages it
