Lecture 7 Flashcards
LBR and Emerin are examples of?
Nuclear envelope proteins
Syne-1 is an example of?
Outer nuclear membrane protein
What regulates liquid liquid phase separation?
Proteins that contain intrinsically disordered regions
What are intrinsically disordered regions?
Parts of the protein that are highly dynamic and can engage in interactions with other proteins
T/F: LLPS depends on concentration of proteins?
Yes, higher the concentration more likely the formation of LLPS
LLPS depends on…
-Concentration of proteins
-Post translational modifications
-Temperature
-pH
-ionic strength
T/F: many LLPS contain RNA and DNA
True
Condensates?
localize a specific protein to a specific compartment
T/F: LLPS can be used to inactivate/activate enzymes?
True
T/F: some condensates are only present under certain conditions?
True
Advantages of bio condensates compared to organelles?
-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
Nuclear Trafficking
NPC can transport things in/out of the nucleus
Modules?
Nucleoporins come together and work in a subcomplex
Example of a Module?
Nup214, Nup358 and Nup88 form a module that helps with the diassembly of the NPC during mitosis
What happens to the NPC during mitosis?
It is disassembled into modules(8) which are reassembled into the NPC after mitosis
What blocks the central gated channel?
Hydrophobic FXF repeats prevent large molecules from diffusing in/out
Different ways the FXF repeats block the central gated channel?
- Oily spaghetti model
- Gummy bear model
We think the central gated channel is block in way between these two
What molecules can passively diffuse through the NPC?
Molecules with diameter of 8nm or less and molecular mass 40kD or less
What molecules can be transported in the NPC via active diffusion?
Particles up to 45nm if they have the proper signal
-Movement can occur in either direction
Signals
Found on large molecules and tell us whether the molecule needs to go in or out of the nucleus
Nuclear localization sequences(NLS)
Signal transport from the cytoplasm to the nucleus
-Permanent
-Can be located anywhere in the polypeptide
Nuclear export signal(NES)?
Signal transport from the nucleus to the cytoplasm
Shuttling Sequences?
Protein can shuttle if it contains both an NLS and NES
Signals for retention
NRS- makes molecule stay in nucleus
CRS- molecule stays in cytoplasm
SV40
-type of NLS signal
-N-KKKRK-C
-Positive charge amino acids important for nuclear localization
What happens if you chane SV40 to KtKRK?
-molecule no longer goes into nucleus
-Suggests we need the positive amino acid
What happens if we flip SV40 C- KRKKK-N
_no nuclear localization
-both the charge and structure of the NLS residues is important for function
Monopartile vs Bipartile NLS
Mono: one cluster of positive amino aicds
Bipartile: two clusters of positive amino acids separated
T/F: nuclear import occurs post translationally?
True
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)
t
Does docking require energy or high temperature?
No
Does translocation from the cytoplasmic side to the nuclear side require energy?
Yes, it must be done at warm temperatures
Does the diassembly of the complex require energy?
Yes must be done in heat
Two subunits of the NLS receptor?
Beta and alpha
T/F: the NLS receptor complex is smaller than 45 nm in diameter?
True since it passes through NPC
Can HIV-1 Caspid fit through NPC?
No it hasa diamter of 60nm
How are large complexes larger than 45nm able to enter the nucleus?
They must have proteins that caninteract with the FG nucleoporins
Ran-GDP vs Ran-GTP concentration
Ran-GDP ; Cytoplasm
Ran-GTP : nucleus
RCC1(GEF)
Found in the nucleus converts Ran-GDP to Ran-GTP
-RCC1 is associated with a chromatin which retains it in the nucleus
RanGAP1
Ran found in the cytoplasm filaemtns mediates hydrolysis of Ran-GTP
Beta subunit of NLS receptor binds?
Ran-GTP
Directionality comes from..
Instability of the NLS complex in the nucleus because once RanGTP binds the complex falls apart
Nup153
Has FXF repeats that bind the beta subunit and help cargo accumulate on the nuclear side (found in the nuclear basket)
NLS-Receptor, importin-beta, NES-Receptor
Transporters that bind only to RanGTP
Adaptors
The NLS alpha subunit is an adaptor protein, there are some transports that do not require these adaptors
Nuclear Export Signals
Many export signals have hydrophobic amino acid residues at specific positions(these are important for the function of the NES)
Old nuclei
-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
What could be the consequences when the NPC permeability barrier breaks down?
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
How can we control the trafficking of individual proteins?
Change concentrations or physological aspects of cells
Control the trafficking of a group of proteins?
increasing the concentration of NLS receptors
control trafficking at the level of the NPC
Change diameter of the central gated channel
How do we get extremely large molecules in/out of the nucleus?
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
How to target something to the inner nuclear membrane using immobilization prep?
- Insert a protein into the ER membrane/outer nuclear membrane
- Protein will then diffuse around and get into the nucleus
- protein gets into the nucleus it gets trapped by binding either to lamins, chromatin of both
How to target something to the inner nuclear membrane using importing?
- Insert the protein into the ER or outer nuclear membrane
- use importins to target the protein to the inner nuclear membrane.
