Lecture 7

Question 1

LBR and Emerin are examples of?

Answer 1

Nuclear envelope proteins

Question 2

Syne-1 is an example of?

Answer 2

Outer nuclear membrane protein

Question 3

What regulates liquid liquid phase separation?

Answer 3

Proteins that contain intrinsically disordered regions

Question 4

What are intrinsically disordered regions?

Answer 4

Parts of the protein that are highly dynamic and can engage in interactions with other proteins

Question 5

T/F: LLPS depends on concentration of proteins?

Answer 5

Yes, higher the concentration more likely the formation of LLPS

Question 6

LLPS depends on…

Answer 6

-Concentration of proteins
-Post translational modifications
-Temperature
-pH
-ionic strength

Question 7

T/F: many LLPS contain RNA and DNA

Answer 7

True

Question 8

Condensates?

Answer 8

localize a specific protein to a specific compartment

Question 9

T/F: LLPS can be used to inactivate/activate enzymes?

Answer 9

True

Question 10

T/F: some condensates are only present under certain conditions?

Answer 10

True

Question 11

Advantages of bio condensates compared to organelles?

Answer 11

-Don’t have to expend energy to make membranes, matrial can be easily transported in/out
-Easy control of when the compartments are present or not
-Easily regulated

Question 12

Nuclear Trafficking

Answer 12

NPC can transport things in/out of the nucleus

Question 13

Modules?

Answer 13

Nucleoporins come together and work in a subcomplex

Question 14

Example of a Module?

Answer 14

Nup214, Nup358 and Nup88 form a module that helps with the diassembly of the NPC during mitosis

Question 15

What happens to the NPC during mitosis?

Answer 15

It is disassembled into modules(8) which are reassembled into the NPC after mitosis

Question 16

What blocks the central gated channel?

Answer 16

Hydrophobic FXF repeats prevent large molecules from diffusing in/out

Question 17

Different ways the FXF repeats block the central gated channel?

Answer 17

1. Oily spaghetti model
2. Gummy bear model
   We think the central gated channel is block in way between these two

Question 18

What molecules can passively diffuse through the NPC?

Answer 18

Molecules with diameter of 8nm or less and molecular mass 40kD or less

Question 19

What molecules can be transported in the NPC via active diffusion?

Answer 19

Particles up to 45nm if they have the proper signal
-Movement can occur in either direction

Question 20

Signals

Answer 20

Found on large molecules and tell us whether the molecule needs to go in or out of the nucleus

Question 21

Nuclear localization sequences(NLS)

Answer 21

Signal transport from the cytoplasm to the nucleus
-Permanent
-Can be located anywhere in the polypeptide

Question 22

Nuclear export signal(NES)?

Answer 22

Signal transport from the nucleus to the cytoplasm

Question 23

Shuttling Sequences?

Answer 23

Protein can shuttle if it contains both an NLS and NES

Question 24

Signals for retention

Answer 24

NRS- makes molecule stay in nucleus
CRS- molecule stays in cytoplasm

Question 25

SV40

Answer 25

-type of NLS signal -N-KKKRK-C -Positive charge amino acids important for nuclear localization

Question 26

What happens if you chane SV40 to KtKRK?

Answer 26

-molecule no longer goes into nucleus -Suggests we need the positive amino acid

Question 27

What happens if we flip SV40 C- KRKKK-N

Answer 27

_no nuclear localization -both the charge and structure of the NLS residues is important for function

Question 28

Monopartile vs Bipartile NLS

Answer 28

Mono: one cluster of positive amino aicds Bipartile: two clusters of positive amino acids separated

Question 29

T/F: nuclear import occurs post translationally?

Answer 29

True

Question 30

T/F: Shuttling proteins can respond to changes relatively fast, we can regulate the distribution of a protein by altering the cell physiology(changing the calcium concentration)

Answer 30

t

Question 31

Does docking require energy or high temperature?

Answer 31

No

Question 32

Does translocation from the cytoplasmic side to the nuclear side require energy?

Answer 32

Yes, it must be done at warm temperatures

Question 33

Does the diassembly of the complex require energy?

Answer 33

Yes must be done in heat

Question 34

Two subunits of the NLS receptor?

Answer 34

Beta and alpha

Question 35

T/F: the NLS receptor complex is smaller than 45 nm in diameter?

Answer 35

True since it passes through NPC

Question 36

Can HIV-1 Caspid fit through NPC?

Answer 36

No it hasa diamter of 60nm

Question 37

How are large complexes larger than 45nm able to enter the nucleus?

Answer 37

They must have proteins that caninteract with the FG nucleoporins

Question 38

Ran-GDP vs Ran-GTP concentration

Answer 38

Ran-GDP ; Cytoplasm Ran-GTP : nucleus

Question 39

RCC1(GEF)

Answer 39

Found in the nucleus converts Ran-GDP to Ran-GTP -RCC1 is associated with a chromatin which retains it in the nucleus

Question 40

RanGAP1

Answer 40

Ran found in the cytoplasm filaemtns mediates hydrolysis of Ran-GTP

Question 41

Beta subunit of NLS receptor binds?

Answer 41

Ran-GTP

Question 42

Directionality comes from..

Answer 42

Instability of the NLS complex in the nucleus because once RanGTP binds the complex falls apart

Question 43

Nup153

Answer 43

Has FXF repeats that bind the beta subunit and help cargo accumulate on the nuclear side (found in the nuclear basket)

Question 44

NLS-Receptor, importin-beta, NES-Receptor

Answer 44

Transporters that bind only to RanGTP

Question 45

Adaptors

Answer 45

The NLS alpha subunit is an adaptor protein, there are some transports that do not require these adaptors

Question 46

Nuclear Export Signals

Answer 46

Many export signals have hydrophobic amino acid residues at specific positions(these are important for the function of the NES)

Question 47

Old nuclei

Answer 47

-central-gated channel may become leaky -function of the NPC becomes compromised and leaky -Large molecules that would be excluded in young nuclei are able to get through the central-gated pore of older nuclei

Question 48

What could be the consequences when the NPC permeability barrier breaks down?

Answer 48

No longer need to use energy to transport large molecules in/out of the nucleus molecules such as transcription factors may enter and transcribe genes that should not be transcribe d

Question 49

How can we control the trafficking of individual proteins?

Answer 49

Change concentrations or physological aspects of cells

Question 50

Control the trafficking of a group of proteins?

Answer 50

increasing the concentration of NLS receptors

Question 51

control trafficking at the level of the NPC

Answer 51

Change diameter of the central gated channel

Question 52

How do we get extremely large molecules in/out of the nucleus?

Answer 52

Ex. Herpesvirus 1. capsid interacts with the inner nuclear membrane forming a vesicle 2. Generation of an envelope of the inner nuclear membrane around the capsid goes into the perinuclear space(vesicle) 3. This vesicle then fuses with the outer nuclear membrane and enters the cytoplasm

Question 53

How to target something to the inner nuclear membrane using immobilization prep?

Answer 53

1. Insert a protein into the ER membrane/outer nuclear membrane 2. Protein will then diffuse around and get into the nucleus 3. protein gets into the nucleus it gets trapped by binding either to lamins, chromatin of both

Question 54

How to target something to the inner nuclear membrane using importing?

Answer 54

1. Insert the protein into the ER or outer nuclear membrane 2. use importins to target the protein to the inner nuclear membrane.