Transport: Going Nuclear Flashcards
Lecture 20
How many ribosomal subunits are transported per passageway between the nucleus and the cytoplasm per minute?
3-7 subunits per minute
Where does transcription and RNA processing occur?
the nucleus
Where does the ribosome get built?
The cytoplasm. The ribosomal subunits are created in the nucleus, then exported to the cytoplasm to build the ribosome.
How far is the nucleus from the endoplasmic reticulum?
Not as far as many believe. They are contiguous.
Are the proteins needed for transcription created in the nucleus or the cytoplasm?
In the cytoplasm.
Are the proteins needed for chromosome replication created in the nucleus or the cytoplasm?
In the cytoplasm.
Do the proteins used for transcription operate in the nucleus or the cytoplasm?
In the nucleus
Do the proteins used for chromosomal replication operate in the nucleus or the cytoplasm?
In the nucleus
What is the distance between the inner and outer nuclear membrane?
The perinuclear space is 20-40 nm. The inner and outer nuclear membranes are contiguous.
What is the perinuclear space?
space between the inner and outer nuclear membranes; contiguous with the ER lumen
How many nuclear core complexes are there in mammalian cells?
Varies among cell types and species. Mammalians can have 3-4 thousand.
What are nuclear pore complexes?
passageways where molecules are transported to or from the nucleus
The inner and outer nuclear membranes are contiguous. What makes them different?
Location, unique compositions of lipids and proteins
How big are nuclear pore complexes (mass)?
120 Megadaltons (MDa)
How many different polypeptides make up nuclear pore complexes?
50-100 different kinds
What do nuclear pore complexes look like in electron micrographs at the cytoplasmic face?
rings with 8-fold symmetry
What do nuclear pore complexes look like in electron micrographs at the nucleoplasmic face?
Fish trap/basket shape, place where molecules go in and out
How many openings are there in a nuclear pore complex?
4 openings
What is the size upper limit to molecules freely diffusing through nuclear pore complexes be?
20 kDa or less
Why does free diffusion of small molecules (smaller than 20 kDa) happen?
There are aqueous diffusion channels within the nuclear pore complex.
How big are nuclear pore complexes (length)?
120 nm
What function does the transporter serve in the nuclear pore complex?
in the middle of the complex, it is where large objects move through
How do ribosomal subunits travel out of the nucleus into the cytoplasm?
The transporter in the nuclear pore complex forces the entire complex to undergo a conformational change.
How does size influence what travels between the nucleus and the cytoplasm?
For small molecules, there is non-selective passive transport.
For larger molecules (greater than 60 kDa), there is greater selectivity, undergoes active transport.
What energy source powers the transportation of large molecules between the nucleus and the cytoplasm?
GTP
What is necessary for a molecule to enter a nucleus?
Nuclear localization signal sequence allows nuclear import.
What does the NLS sequence do?
The nuclear localization sequence allows a molecule larger than 60 kDa to enter the nucleus of a cell.
What is necessary for a polypeptide larger than 60 kDa to enter the nucleus?
NLS sequence and GTP
Describe the experiment that proved the NLS is necessary for large proteins to move into the nucleus.
The polypeptide was fluorescently labeled and the presence or absence of the NLS determined whether the polypeptide ended up in the cytoplasm or the nucleus.
What constitutes the NLS sequence?
No consensus, but generally prolines and basic amino acids (lysines and arginines) are present.
What 3 kinds of amino acid residues are generally present in the NLS?
prolines, lysines, and arginines
How wide can the central channel of the nuclear pore complex dilate to (active transport)?
about 30 nm
Describe the experiment that helped discover the size limits of molecules traveling through the nuclear pore complex. What was discovered?
Used gold particles of different sizes, coated them with proteins that had nuclear localization signals, and saw what sizes of particles were entering the nucleus
Viewed with electron microscopy
Discovered that particles with diameters of about 30 nm can enter via the NPC
Why were gold particles used to discover the size range of molecules traveling in the nuclear pore complex?
Because gold particles deflect electron beams, causing them to appear dark in electron microscopy.
Name 2 examples of illnesses caused by virions using the nuclear pore complexes. Describe them.
Hepatitis B is caused by virions moving through the NPC.
CMV is caused by a virion sitting on top of the NPC and releasing nucleic acids through the complex.
How do you turn the Ran G protein off? What does the “off” state look like?
To turn off, converting to a G-GDP, use a GAP (GTPase activating protein)
How do you turn the Ran G protein on? What does the “on” state look like?
To turn on, converting to a G-GTP, use a GEF (Guanine Nucleotide Exchange Factor).
Describe the nuclear importation process of a protein.
- Cargo with NLS must bind to importin, a soluble protein, at the alpha subunit.
- Importin’s beta subunit binds to proteins in the transporter, moving between polypeptides WITHOUT nucleotide hydrolysis.
- Cargo complex enters the nucleus.
- Cargo complex interacts with Ran-GTP, causing the importin to release the cargo.
- Importin-Ran-GTP complex is formed, which exits the nucleus, returning to the cytoplasm.
- GAF in the cytoplasm converts Ran-GTP to Ran-GDP, causing importin to disassociate. Importin is free to bind more cargo.
What four things are needed for nuclear importation?
Cargo with an NLS sequence
Importin
Ran-GTP and Ran-GDP
Describe importin.
Receptor of the cargo with an NLS sequence traveling from the cytoplasm into the nucleus
Soluble (NOT transmembrane) protein
Dimer, with an alpha and beta subunit
Where is importin before importation begins?
in the cytoplasm (not embedded in the nuclear membrane)
What does the alpha subunit of importin do?
Binds to the cargo
What would happen if a GAP were not present in the cytoplasm during nuclear importation?
Ran-GTP would never convert into Ran-GDP, so the importin would not leave the importin-Ran-GTP complex. No more cargo could enter the nucleus.
What does the beta subunit of importin do?
Binds to the proteins in the nuclear pore complex’s transporter
Importin is a dimer. What do each of the subunits do?
The alpha subunit binds to the cargo; the beta subunit binds to proteins in the transporter of the NPC.
What would happen if Ran-G protein were not present in the nucleoplasm during nuclear importation?
The importin could not disassociate from the cargo in the nucleoplasm.
Describe the experiment that explained the process of nuclear importation.
In vitro reconstitution
A special detergent that solubilized (poked holes in) membranes were used on cells, allowing researchers to diffuse different molecules.
First diffused a sample of isolated cytosol, mixed with fluorescently-labeled BSA protein with an NLS sequence to see if it would enter the nucleus.
Second, did same experiment with cytosol, BSA, and Ran. Discovered that with it would not enter the nucleus.
Third, used Ran, BSA, and importin; discovered the protein entered the nuclei.
Fourth, used BSA and importin without Ran: discovered the ring around the nucleus lit up, but it did not enter the nucleus.
What is minimally necessary for a protein to enter the nucleus?
Ran
Importin
Protein with NLS
What happens if a protein with an NLS and importin undergoes importation without Ran?
Without Ran, the importin cannot separate from the cargo, so the cargo never enters the nucleoplasm. The complex remains at the surface of the nucleus. The cycle is interrupted.
Why is nuclear import unidirectional?
Import is down a concentration gradient. The concentration of the cargo complex is high in the cytoplasm relative to the nucleus.
What is active about nuclear importation? What’s passive?
Active - uses GTP hydrolysis
Passive - goes down a concentration gradient
What direction is nuclear import?
Unidirectional from the cytoplasm to the nucleus, down a concentration gradient
Nuclear import is down a concentration gradient. What establishes the gradient?
The Ran GTPase cycle. As soon as the cargo complexes enter the nucleus, they break apart due to Ran-GTP.
Why is there a concentration gradient for Ran-GTP?
Ran GTP is formed in the nucleus because Ran GEF is associated with chromatin (held in the nucleus).
Where is Ran GTP formed? Why?
In the nucleus, because the Ran-GEF is associated with chromatin and converts Ran-GDP to Ran-GTP.
Where is Ran-GEF during nuclear import?
in the nucleus, associated with chromatin
Where is Ran-GAP during nuclear import?
in the cytoplasm
What is the nucleolus?
region in the nucleus that transcribes DNA into rRNA; considered the ribosome factory of the cell
How many nucleoli are in the nucleus?
depends on the state of the cell, type of cell, species; could be one or a dozen
What do the light regions of an electron micrograph of the nucleolus show?
Fibril regions, where transcription of ribosomal DNA occurs
Where does the transcription of ribosomal DNA occur?
in the fibril regions of the nucleolus in the nucleus
Where is mRNA produced?
at the fibril regions of the nucleolus in the nucleus
Where is rDNA transcribed?
in the nucleolus
What do the 2 mechanisms for nuclear export transport?
Ran-dependent mechanism is used to transport ribosomal subunits, tRNA, and proteins.
Ran-independent mechanism is used to transport mRNA.
What do the 2 mechanisms for nuclear transport require?
The first mechanism requires a Nuclear Export Signal (NES), a Ran gradient, and exportin.
The second mechanism requires an mRNA exporter protein, but doesn’t require Ran or exportin.
Describe the mechanism for the nuclear export of ribosomal subunits, tRNA, and proteins.
- Cargo with NES sequence binds to exportin receptor and Ran-GTP, forming a trimeric complex in the nucleus.
- The trimeric compelx passes through the nuclear pore complex
- A Ran-GAP hydrolyzes the Ran-GTP into Ran-GDP, causing the complex to break apart.
- Ran-GDP re-enters the nucleus and Ran-GTP is regenerated by Ran-GEF.
Describe the mechanism of nuclear export of mRNA.
- mRNA molecules are coated with mRNA exporter proteins.
- mRNA exporter protein complex exits the nucleus via the nuclear pore complex.
What does the nuclear export of mRNA depend on?
ATP
Ribosomal subunits, mRNA, tRNA, and proteins all exit the nucleus. Which mechanisms do they use?
Ribosomal subunits, tRNA, and proteins all use the mechanism with the NES, Ran gradient, and exportin.
mRNA is the only molecule that uses mRNA exporter proteins and does not require exportin or Ran GTPase.
Is the nuclear pore complex required for nuclear export?
No. There is a mechanism where a big object can be wrapped in the inner nuclear membrane, fused with the outer nuclear membrane, and then released into the cytoplasm without a NPC.
What is an example of molecules that do not exit the nucleus via nuclear pore complexes?
Herpes virus capsids are 125 nm, much too large to exit through NPCs. They are engulfed by the nuclear envelope and eventually released into the cytoplasm.
What is the nuclear envelope composed of?
inner and outer nuclear membranes
What is the outer nuclear membrane continuous with?
The ER
What is the perinuclear space continuous with?
the lumen of the ER
What roles does Ran-GTP play in nuclear import? In export?
In import, Ran-GTP promotes the release of importin from the cargo.
In export, the Ran-GTP promotes the binding of the exportin, the cargo, and itself. All are released together.
Using the marvels of genetic engineering you express in a fungal cell, in order from N-
terminus to C-terminus, a nuclear localization sequence (NLS) and a nuclear export sequence
(NES). Where in the cell would you expect to find this engineered protein?
A. In the cytoplasm
B. In the nucleoplasm
C. In the perinuclear space
D. Answers (A) and (B)
E. Answers (B) and (C )
D. A and B.
NLS and NES do not get cleaved. They will continuously cycle.
Co-translational insertion of polypeptides into the lumen of the perinuclear space _________. (from old test).
A. never occurs
B. requires translocons
C. requires GTP hydrolysis
D. requires nuclear pore complexes
E. Answers (B) and (C).
E. B and C
Perinuclear space is the ER lumen (they are continuous).
Using the marvels of genetic engineering you express in a mammalian cell, in order from N-terminus to C-terminus, a signal sequence that binds signal recognition particles (SRPs), a nuclear localization signal (NLS), a signal-anchor
(start-transfer) sequence, and a KKXX sequence. Where in the cell would you expect to find the greatest concentration of this engineered protein? (from 2019)
A. In the cytosol
B. Spanning the basolateral domain of the plasma membrane
C. In the lumen of the endoplasmic reticulum
D. In the nucleoplasm
E. Spanning the outer nuclear membrane
E. Spanning the outer nuclear membrane
The outer nuclear membrane and the ER membrane are continuous.
SRP - rER translation - leads to SRP on ER or outer nuclear membrane
NLS - makes it to nucleus
Signal-anchor - transmembrane protein
KKXX - transmembrane, will be retained in ER membrane (AKA the outer nuclear membrane)
