The Cell-38

Question 1

What do membranes act sa?

Answer 1

Walls of a house

Question 2

Do all eukaryotic cells look the same?

Answer 2

No, they can look extremely different.

E.g. RBC, breast cancer cells, osteocyte, cork cells, mold hyphae cells, orange pepper cells, diatoms, heart muscle cells

Question 3

Do eukaryotic cells function the same?

Answer 3

Yes, they function similarly.

Life is made of the same types of molecules in all known organisms. They all have a basic set of membrane-enclosed organelles (compartmentation).

Question 4

What needs to move within eukaryotic cell space?

Answer 4

Proteins. Protein synthesis starts in the cytosol, but needs to move around the cell.

Eukaryotic cells are very crowded.

Proteins that live in organelles or are secreted out of the cell must be sorted (sent) to the correct compartment using sorting signals.

Question 5

What is the roles of organelles?

Answer 5

Nucleus- site of DNA and RNA synthesis.
Cytoplasm- contains cytosol and organelles.
Cytosol- 50% of cell volume, site of protein synthesis and many metabolic pathways.
ER- 50-60% of cell membrane, start point of secretory pathway.
Golgi apparatus- 10% of cell membrane, important for sorting and modifying proteins and lipids passing through it.
Lysosomes- 1% of cell volume, multiple ‘suicide’ bags for digestion of materials.
Mitochondria/chloroplasts- 25% of cell volume, generate ATP.
Peroxisomes- 1% of cell volume, multiple sites of oxidative reactions.
Plant vacuoles- 90% of cell volume, for turgor or protein storage/degradation.

Question 6

What organelles are in the cytoplasm?

Answer 6

ER, lysosomes, mitochondria, chloroplasts, peroxisomes and plant vacuoles.

Question 7

What is compartmentation?

Answer 7

Eukaryotic cells have a basic set of membrane-enclosed organelles.

Question 8

Who were the 4 Nobel Prizes awarded to for Protein Transport?

Answer 8

1999- Gunter Blobel. For the discovery that proteins have intrinsic signals that govern their transport and localisation in the cell.
2013- Jim Rothman, Randy Schekman and Thomas Sudhof. For the discoveries of machinery regulating vesicle traffic, a major transport system in our cells.

All for Physiology or Medicine.

Question 9

Why does synthesis of proteins begin in the cytosol?

Answer 9

That is where ribosomes are located.

Question 10

What is gated transport?

Answer 10

Import into and export out of the nucleus.

From the nucleus to the cytosol and vice versa.

Question 11

What is protein translocation?

Answer 11

Protein import into the ER, plastids, mitochondria or peroxisomes.

Question 12

What is vesicular transport?

Answer 12

Secretion along the organelles of the secretory pathway.

E.g. ER to peroxisomes.
ER to golgi and vice versa.
Golgi to other organelles and vice versa.

Question 13

How do proteins identify where to go?

Answer 13

Proteins that do not reside in the cytosol need sorting signals to reach their correct destination.

Question 14

Where are sorting signals?

Answer 14

They are part of the protein.

Question 15

What are the types of sorting signals on the protein?

Answer 15

Short peptides at the N- or C-termini
-can be removed after use or kept on if needed again (for nuclear transport).

3-D domains (secondary/tertiary structure)
-e.g. for transport to lysosomes

Other molecules attached to the protein (post-translational modifications)
-sugars (e.g. lysosomes)
-lipids

Question 16

What happens to sorting signals?

Answer 16

-They are recognised by specific receptors within the cell.
-Triggers transfer of the protein to the correct destination.
-Every organelle uses different receptors and different sorting process.
-Can be removed after use or kept on if needed again.

Question 17

What happens if there is missorting of signals?

Answer 17

The cell is in big trouble.

Several diseases result from the mis-sorting of proteins (traffic jams).

Question 18

How do proteins and other macromolecules move between the cytoplasm and the nucleus?

Answer 18

Via large aqueous nuclear pore complexes (NPC).

Question 19

Why does gated transport into the nucleus need to occur?

Answer 19

Because the nucleus is the site for DNA and RNA localisation.

Question 20

What is a key feature of nuclear pores?

Answer 20

8 fold symmetry.

Has basket-like fibrils.

A mammalian nuclear envelope contains 3000-4000 NPCs.

Question 21

How large are NPCs?

Answer 21

Each NPC is 125,000 kDa. This is 30x bigger than a ribosome (around 3,300 kDa).

Is made up of many copies (16, or multiples of 16) of around 30 different nucleoporins.

Nucleoporins line the channel, the nuclear basket and the cytosolic fibrils.

Question 22

How fast are NPCs?

Answer 22

1000 macromolecules per second.

Question 23

How does size affect diffusion?

Answer 23

Small molecules (5 kDa or less) diffuse extremely fast (almost free flow).

Proteins of 20-40 kDa diffuse more slowly.

Question 24

How does size affect active transport?

Answer 24

Proteins >40 kDa cannot move across, they carry nuclear localisation or export signals.

Question 25

How many human proteins are nuclear?

Answer 25

22%. Therefore, must be imported from the cytosol. Ribosome protein subunits need to enter the nucleus to bind rRNA, and exit the nucleus to their cytosolic destination.

Question 26

How is the diffusion barrier caused?

Answer 26

By the unstructured regions of channel nucleoporins rich phenylalanine-glycine (FG) repeats forming a tangled network (mesh or matrix). This blocks the passive diffusion of large molecules (e.g. ribosomes).

Question 27

What are Nuclear Localisation Signals (NLS)?

Answer 27

NLS are rich in lysine and arginine (+charged), and can be in any position of the passenger (=cargo) protein. As long as they are exposed to the surface of the protein. NLS are recognised by nuclear import/export receptors.

Question 28

How are NLS recognised?

Answer 28

By a family of cytosolic nuclear import receptors (importins or karyopherins). Importins an bind to different types of cargo proteins or adaptor proteins. They bind and detect receptor. Each member being responsible for NLS- a set of cargo molecules (or sometimes a cargo adaptor)

Question 29

What forms the mesh?

Answer 29

Nucleoporins with FG repeats form the mesh.

Question 30

Can diffusion still occur if there is a diffusion barrier?

Answer 30

No, large molecules cannot passively diffuse. So active transport takes place instead.

Question 31

What happens as a result of mesh formed?

Answer 31

Import receptors have low affinity binding sites for the FG repeats. FG repeats in the cytosolic fibrils help recruit the receptors and bind cargo. This complex then travels through the pore mesh through transient interactions. The receptor disengages from the cargo protein on the other side. And is released so transport happens.

Question 32

How is nuclear import active?

Answer 32

Moving certain proteins inside the nucleus creates different protein pools in the nucleoplasm (interior of nucleus) compared the the cytoplasm. This type of nuclear transport creates order in the cell. The energy for this type of transport comes from GTP via the GTPase-Ran

Question 33

What is order?

Answer 33

Energy required to maintain it (this type of nuclear transport).

Question 34

What is GTPase-Ran and how does it work?

Answer 34

Importin binds to a GTPase 'switch' protein (Ran). GTPase 'switch': -hydrolyses GTP to GDP (binds both) -conformation is different depending if it bound to GTP or GDP -different conformation = different activity

Question 35

What does GTPase-Ran provide?

Answer 35

Both the free energy and the directionality for nuclear transport. The net import of cargo is driven by two conformational states of the GTPase-Ran. This depends on whether Ran binds GDP or GTP.

Question 36

What does GDP/GTP-Ran have a conformation of and where is this state?

Answer 36

GDP-Ran has conformation (A) and is in this state only in the cytosol. GTP-Ran has conformation (B) and is in this state only in the nucleus.

Question 37

What is GTPase-Ran regulated by?

Answer 37

GAP and GEF. The asymmetric distribution of GTP-Ran (most in in the nucleus) and GDP-Ran (most in the cytosol) is determined by the different location of proteins that can 'switch' Ran on/off.

Question 38

What is GAP and GEF?

Answer 38

GAP- GTPase activating protien) promotes hydrolysis of GTP to GDP). Only in the cytosol. GEF- guanine nucleotide exchange factor (exchanges GDP with GTP). Only in the nucleus. GAP and GEF have different locations, different affinities, accumulation and protein pools.

Question 39

Give a summary of nuclear import (active transport)

Answer 39

GTPase activating protein- GAP ensures that GDP-Ra is generated, and in this state Ran falls off the importin. Facilitated transport via low affinity binding or importin to FG repeats. Guanine nucleotide exchange factor- GEF ensures that Ran-GTP is generated and this prefers to bind to importin, displacing the imported protien.

Question 40

Why is is NLS not cleaved after import? is this common?

Answer 40

-It is often needed again. This is because many nuclear proteins constantly cycle to the cytosol, so they must be repeatedly imported. -Re-import must also happen after every mitosis when the nucleus reforms. -The NLS may be internal and part of a functional domain, therefore uncleavable. -When proteins go into the nucleus, they might loose their signal. -During cell division, nuclear content may dissapear. No, this is unlike other signal peptides for protein sorting.

Question 41

What are exportins? How do they work?

Answer 41

Exportins are nuclear export receptors. They see nuclear export signals (NES). They bind the NES and the FG-nucleoporins in the channel and filaments, to move the cargo to the cytosol. They use Ran in reverse (GDP-Ran releases cargo in the cytosol.

Question 42

How can subcellular organelles be visualised in vivo?

Answer 42

Using dyes or flourescent proteins e.g. GFP.

Question 43

Is nucleo-cytoplasmic trafficking reversible?

Answer 43

Yes, technique which can be sued to visualise organelles.

Question 44

What are other roles of NPCs?

Answer 44

They also control genome regulation and ageing. Nup98 activates genes in the context of acute myeloid leukemia. Some Nups have a very long half-life (several years!), so they deteriorate with age and affect cellular health.