Section 6B: Protein sorting between cytoplasm and nucleus

Question 1

Nuclear-Cytosol Transport (from nucleus into cytosol):

Answer 1

• 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

Question 2

Where are ribosomes made?

Answer 2

they are made in the nucleus and immediately need to leave nucleus to go into the cytosol

Question 3

Nuclear-Cytosol Transport (from cytosol to nucleus):

Answer 3

• ribosomal proteins for
  assembling the ribosome
• DNA polymerase
• DNA repair proteins
• RNA polymerase
• Transcription factors
• mRNA processing enzymes
• Histones
• etc

Question 4

Molecules are diverse:

Answer 4

• Some extremely large
  (ribosome)
• Some of very small
  (small proteins)
• Some even smaller
  (ATP)

Question 5

What is the whole point of the nucleus?

Answer 5

• to surround DNA to prevent/allow things to go in and out
• wrong thing entering DNA will cause DNA damage

Question 6

The Nuclear Envelope

Answer 6

• 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

Question 7

What is a Lamina?

Answer 7

nuclear skeleton underlying inner bilayer - gives shape

Question 8

What is a Nuclear Pore?

Answer 8

“hole” that crosses both inner and outer bilayers
- number of define proteins (nuclear porins) that fit together to form a passageway

Question 9

The Nuclear Pore Complex (NPC)

Answer 9

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

Question 10

< 5kDa

Answer 10

free diffusion

Question 11

5 - 60 kDa

Answer 11

diffusion decreases with size

Question 12

> 60 kDa

Answer 12

active transport needed

Question 13

How do you keep something small from coming in and out?

Answer 13

• 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

Question 14

Nuclear pore complex is a _______ gate

Answer 14

selective
- some proteins need to be in the nucleus and others out

Question 14

Nuclear pore complex forms a gate that controls transport

Answer 14

• 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

Question 15

What targets a protein to the nucleus?

Answer 15

Nuclear Localization Signal (NLS)

Question 16

What is the job of the Nuclear Localization Signal (NLS)?

Answer 16

it carries a code inside a protein to tell it what to do

Question 17

What happens when you artificially attach a nuclear localization sequence to GFP?

Answer 17

it will drag the whole thing into the nucleus

Question 18

Nuclear Import Receptors

Answer 18

• 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

Question 19

What do Nuclear Import Receptors do?

Answer 19

bind to cargo (e.g. proteins being transported into the nucleus) and bridge these into the NPC

Question 20

Nuclear Export Signals and Receptors

Answer 20

• 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)

Question 21

What causes directionality?

Answer 21

• Active Transport
• Low concentration from cytosol to high concentration to nucleus)

Question 22

How do certain proteins accumulate in the nucleus?

Answer 22

There has to be something (GTPase) that
causes ACTIVE transport and
establishes DIRECTION of
transport

Question 23

GTPase “switches”

Answer 23

1. ON state: bound to GTP
2. OFF state: bound to GDP

Question 24

What is the protein to turn GTPase ON?

Answer 24

GEF

Question 25

What is the protein to turn GTPase OFF?

Answer 25

GAP

Question 26

Ran GTPase regulates _______ of nuclear transport

Answer 26

directionality

Question 27

Ran GTPase and the nuclear transport cycle

Answer 27

- Nuclear Import - Nuclear Export

Question 28

Nuclear Import:

Answer 28

- 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”

Question 29

Ran is bound to GTP in the nucleus and GDP in the cytosol, what does this mainatin?

Answer 29

This maintains GRADIENT

Question 29

Nuclear Export:

Answer 29

- 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”

Question 29

T cells are immune cells that are activated during infection and inflammation

Answer 29

- 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

Question 29

RanGTP binding blocks a protein loop required for binding to cargo

Answer 29

- 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

Question 29

Nuclear transport by Ran-GTP and Ran-GDP gradient: Summary

Answer 29

- 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

Question 29

Nuclear Import Chart

Answer 29

- 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)

Question 29

Nuclear Export Chart

Answer 29

- 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)

Question 30

GTP hydrolysis in the cytosol deposits...

Answer 30

export cargo in the cytosol

Question 30

Access to the transport machinery can be turned ON and OFF

Answer 30

e.g. infection in blood - only turn on immune cells when there is an infection - protein changes nuclear or cytosolic localization

Question 31

Control nuclear import during T cell activation

Answer 31

- 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

Question 32

What happens when Calcineurin is stopped?

Answer 32

T cells no longer have the ability to destroy cells

Question 32

What does low Ca2+ mean?

Answer 32

it means infection has cleared

Question 32

Low Ca2+ is in _______ T cell, High Ca2+ is in _______ T cell

Answer 32

resting, activated