Section 6B: Protein sorting between cytoplasm and nucleus Flashcards
Nuclear-Cytosol Transport (from nucleus into cytosol):
- processed mRNA (has to be in nucleus when first generated)
- enzymes come and read DNA code to make RNA (transcription)
-tRNAs - assembled ribosomes
- transcriptional regulators (has to let massive things go through nuclear envelope
Where are ribosomes made?
they are made in the nucleus and immediately need to leave nucleus to go into the cytosol
Nuclear-Cytosol Transport (from cytosol to nucleus):
- ribosomal proteins for
assembling the ribosome - DNA polymerase
- DNA repair proteins
- RNA polymerase
- Transcription factors
- mRNA processing enzymes
- Histones
- etc
Molecules are diverse:
- Some extremely large
(ribosome) - Some of very small
(small proteins) - Some even smaller
(ATP)
What is the whole point of the nucleus?
- to surround DNA to prevent/allow things to go in and out
- wrong thing entering DNA will cause DNA damage
The Nuclear Envelope
- double, continuous bilayer
- outer membrane continuous with ER
- Lamina
- Nuclear pore
- controls bidirectional transport between nucleus and cytosol
- 3000-4000 pores/nucleus in mammalian cells
- 5000 macromolecules in both directions
What is a Lamina?
nuclear skeleton underlying inner bilayer - gives shape
What is a Nuclear Pore?
“hole” that crosses both inner and outer bilayers
- number of define proteins (nuclear porins) that fit together to form a passageway
The Nuclear Pore Complex (NPC)
Disordered region of channel
- Made up of proteins (nucleoporins) that are disordered – lack secondary structure
- Forms a “barrier” that larger
molecules (>60 kDa) cannot
pass without assistance
- Large molecules thus require active transport involving:
1. recognition of nuclear
signal
2. Interaction with cytosolic
fibrils
3. Passage through NPC
< 5kDa
free diffusion
5 - 60 kDa
diffusion decreases with size
> 60 kDa
active transport needed
How do you keep something small from coming in and out?
- opening is full of proteins that likes to stick together thus, makes it hard for other things to go into complex
- but allow giant things to go through
Nuclear pore complex is a _______ gate
selective
- some proteins need to be in the nucleus and others out
Nuclear pore complex forms a gate that controls transport
- prevents the free movement of molecules
- large molecules cannot diffuse through without Active Transport
- intermediate molecules can diffuse through, but very slowly
- super small molecules can diffuse through easily
- Central nucleoporins have unstructured domains that line the pore
- Creates a meshwork that slows diffusion
- Proteins >60 kDa must be actively transported in and out of the nucleus
- Nuclear pore complex is a selective gate: some proteins need to be in the nucleus and others out
What targets a protein to the nucleus?
Nuclear Localization Signal (NLS)
What is the job of the Nuclear Localization Signal (NLS)?
it carries a code inside a protein to tell it what to do
What happens when you artificially attach a nuclear localization sequence to GFP?
it will drag the whole thing into the nucleus
Nuclear Import Receptors
- looks for Nuclear Localization Signal (NLS)
- be able to bind to these fibre that come out of the NPC
- NPC often does not interact with transport “cargo” protein directly; they need an intermediate called Nuclear Import Receptors
What do Nuclear Import Receptors do?
bind to cargo (e.g. proteins being transported into the nucleus) and bridge these into the NPC
Nuclear Export Signals and Receptors
- Nuclear export also occurs through signal peptide sequences: nuclear export signal
- Nuclear export signal is recognized by nuclear export receptors (aka karyopherins)
- Example function: export pre-assembled ribosomes and mRNA (these are proteins that patrol the nucleus)
What causes directionality?
- Active Transport
- Low concentration from cytosol to high concentration to nucleus)
How do certain proteins accumulate in the nucleus?
There has to be something (GTPase) that
causes ACTIVE transport and
establishes DIRECTION of
transport
GTPase “switches”
- ON state: bound to GTP
- OFF state: bound to GDP
What is the protein to turn GTPase ON?
GEF
What is the protein to turn GTPase OFF?
GAP
Ran GTPase regulates _______ of nuclear transport
directionality
Ran GTPase and the
nuclear transport cycle
- Nuclear Import
- Nuclear Export
Nuclear Import:
- In the cytoplasm, Ran-GDP does not bind to nuclear import receptors,
which allows binding to proteins destined for the nucleus - Once past the Nuclear Pore Complex (NPC), the nuclear import
receptors bind to Ran-GTP (in the nucleus). - Ran-GTP forces the nuclear import receptors to release their protein
cargo - Nuclear import receptors bound to Ran-GTP return to cytosol “empty”
Ran is bound to GTP in the nucleus and GDP in the cytosol, what does this mainatin?
This maintains GRADIENT
Nuclear Export:
- In the nucleus, Ran-GTP binds to nuclear export receptors, which allows
binding to proteins destined for the cytosol. - Once past the Nuclear Pore Complex (NPC), the nuclear export
receptors bind to Ran-GDP (in the cytosol, due to Ran-GAP). - Ran-GDP forces the nuclear export receptors to release their protein
cargo. - Nuclear export receptors bound to Ran-GDP return to nucleus “empty”
T cells are immune cells that are activated during infection and inflammation
- destroy infected cells
- Once activated, T-cells need to “turn on” certain genes
- NF-AT is a transcription factor that turns on genes when T cells
are activated - NF-AT hangs out in cytosol but moves into nucleus once T cells are
activated
RanGTP binding blocks a protein loop
required for binding to cargo
- binding of Ran GTP to the nuclear import receptor causes a change in shape of the nuclear import receptor, changing conformation that no longer makes it possible for the cargo to bind
- cargo releases
Nuclear transport by Ran-GTP
and Ran-GDP gradient: Summary
- There is a Ran-GTP/GDP gradient: because Ran GEF is in the nucleus and Ran GAP is in the cytosol
- Ran-GTP is high in the nucleus and low in the cytosol
- Ran-GDP is low in the nucleus and high in the cytosol
- Ran-GTP binds to nuclear import receptors to deposit cargo proteins in the nucleus
- Ran-GTP induces binding of nuclear export receptors to cargo proteins
- GTP hydrolysis in cytosol deposits export cargo in the cytosol
Nuclear Import Chart
- Transport Pore: Nuclear Pore Complex (NPC)
- Sequence recognized on
CARGO proteins: Nuclear Localization
Sequence (NLS) - Transport receptors: Nuclear Import
Receptor - Effect of binding Ran-GTP
(in nucleus): Nuclear Import
Receptor lets go of
cargo (into nucleus) - Effect of Ran-GDP
(in cytosol): Nuclear Import
Receptor can bind to
cargo (in cytosol)
Nuclear Export Chart
- Transport Pore: Nuclear Pore Complex (NPC)
- Sequence recognized on
CARGO proteins: Nuclear Export Sequence (NES) - Transport receptors: Nuclear Export
Receptor - Effect of binding Ran-GTP
(in nucleus): Nuclear Export
Receptor can bind to cargo (in the nucleus) - Effect of Ran-GDP (in cytosol): Nuclear Export Receptor lets go of cargo (in cytosol)
GTP hydrolysis in the cytosol deposits…
export cargo in the cytosol
Access to the transport machinery can be turned ON and OFF
e.g. infection in blood
- only turn on immune cells when there is an infection
- protein changes nuclear or cytosolic localization
Control nuclear import during T
cell activation
- T cells are immune cells – responsible for inflammatory response
- Normally inactive until expose to inflammatory signals or antigens
- When inactive – NF-AT, a transcription factor is cytosolic (phosphorylated) to keep inflammation genes off
- When activated – NF-AT is dephosphorylated to move to the
nucleus – drives gene expression of inflammatory genes - Phosphorylation and dephosphorylation are on-off switches to expose nuclear export and nuclear import signals, respectively
- Low Ca2+ is in resting T cell, High Ca2+ is in activated T cell
- Low Ca2+ means infection has cleared
- When Calcineurin is stopped, it no longer has the ability to destroy cells
What happens when Calcineurin is stopped?
T cells no longer have the ability to destroy cells
What does low Ca2+ mean?
it means infection has cleared
Low Ca2+ is in _______ T cell, High Ca2+ is in _______ T cell
resting, activated
