Lecture 12 - Intracellular compartments and protein sorting Flashcards
Advantages of sub cellular compartments
Allows different ‘factories’ within the cell
Incompatible metabolic processes can go on next to each other
e.g. pH, ion concentration
Sub cellular compartmentalisation has driven
Evolution
Organelles have (4)
- Specific protein and lipid composition
- Specific structure
- Specific number
- Specific arrangement
Organelle number is up or downregulated depending on (2)
The environment
The metabolic needs of the cell
The Golgi can fragment during cell division
Into two structures to be distributed to mother and daughter cells
Membrane contacts allow
Communication between organelles and cells
Tether proteins
Aid membrane fusion
Exchange factors
Involved in cell signalling pathways
An animal cell contains
10 billion protein molecules
10,000 different kinds
Almost all proteins are synthesised in the
Cytoplasm
Most proteins need to be transported
To the correct location to be functional
Phospholipid bilayers allow
The formation of compartments
Structure of a phospholipid
- Hydrophobic tails
2. Hydrophilic head
Structure of the hydrophobic tail of a phospholipid
Non Polar
One straight hydrocarbon tail
One bent hydrocarbon tail - a cis C=C double bond
Structure of the hydrophilic head of a phospholipid
Polar
Hydrophobic tails are bonded to a Glycerol
Glycerol is bonded to a Phosphate
Phosphate is bonded to a variable residue
e.g. choline, ethanolamine
Phospholipids are amphipathic/amphiphilic
Containing both polar and non polar parts
Lipophilic
Fat loving, non polar, fatty acid
The variable residue bonded to the polar head group of a phospholipid provides
Different properties
All lipid molecules in the cell are
Amphipathic
The C=C ‘kink’ in the phospholipid tail allows for
Membrane fluidity
The length of the phospholipid tail also allows for
More electrostatic interactions between other phospholipids (more fluidity)
Cholesterol
Helps maintain membrane fluidity and flexibility
Structure of cholesterol
Polar head group attached to
Rigid steroid rings attached to
Non polar hydrocarbon tail
Cholesterol stabilises membranes
By interacting with fatty acids (slipping inbetween) the phospholipids
Without cholesterol in the membranes what would happen? (2)
When temperatures are increased the membranes could burst
When temperatures are decreased phospholipids could crystallise
Cholesterol has an important role in
Permeability and mobility of membranes
Micelle
Lipid molecules that arrange themselves in a spherical form in aqueous solutions in a single layer
Vesicle
A large structure consisting of liquid enclosed by a lipid bilayer
The two fatty acid tails in a phospholipid cause it to form a
Bilayer
Why do phospholipids form enclosed compartments?
Exposure of hydrocarbon tails is energetically unfavourable
A phospholipid with a single tail will form
Micelles (single layer)
Lipid molecules in a bilayer will diffuse laterally at around
2um/sec (very dynamic)
Phospholipid translocators
Enzymes that catalyse the rapid ‘flip flop’ of phospholipids from one side of the bilayer to the other
Interactions between lipids and proteins in the bilayer
Form micro domains
Influences membrane and protein properties
e.g. Signalling processes, micro domain formation
Micro domains + cholesterol are called
Rafts
Rafts
‘Swim’ in the lipid bilayer
What do rafts do?
Islands for signalling
How do cells move proteins between compartments? (3)
- Gated transport
- Transmembrane transport
- Vesicular transport
A type of organelle transport that has selective gates that actively transport large molecules and diffuse small molecules is?
Gated transport
Example of gated transport
Transport into the nucleus
A type of organelle transport that unwinds and pulls specific proteins through the membrane
Transmembrane transport
Example of transmembrane transport
Transport in mitochondria, plastids, peroxisomes, ER
A type of organelle transport that has membrane enclosed vesicles, that fuse with the membrane of another organelle to release cargo
Vesicular transport
Example of vesicular transport
ER, Golgi, secretory vesicles, lysosomes, late endosomes, early endosomes
Each of the transport systems has
Particular proteins associated with it
Proteins are made in
The cytosol on ribosomes
Proteins are transported to specific places depending on their
‘Labels’ (sorting signals)
Sorting signals are
Amino acid ‘labels’ at the end of a polypeptide that tell them where to go
They have specific physio-chemical properties
Signal peptidases
Remove the sorting signal after transport
Some signals are
Spaced out along the protein, coming together to form a 3d ‘signal patch’
Recognition based on protein-protein interactions forms the basis of
Most cellular processes
Positively charged amino acids in a protein can be a signal for
Nuclear import
Signals can be for
Import or Export
Sorting signals are usually at the
N terminus
Which end of a protein exits the ribosome first?
N terminus
Signals are usually at the beginning of a protein (N terminus)
Because the folding is inhibited otherwise the signal will be folded into the structure
How can you identify sorting signals? (4)
- Identify minimal sequences that are responsible for targeting a specific protein
- Fuse that sequence to a reporter gene (GFP)
- Mutate single amino acids within the sequence
- This will determine which amino acids are integral to targeting (as the protein will remain in the cytoplasm and not be transported)
Gated transport into the nucleus is through
Nuclear pores
Structure of a nuclear pore
30 proteins make a ‘basket’
Only 5 types of proteins:
- Annular subunits (central)
- Lumenal subunits (TM)
- Ring subunits (faces)
- Fibrils (FG repeats)
- Nuclear basket
Unstructured proteins inside a nuclear pore form a tangled network that
Resists movement
Molecules less than 5kDa can move
Freely through a nuclear pore
Molecules greater than 60kDa
Need to be actively transported
Fibrils
Seal the pore
Grab proteins that should go into the nucleus
What is transported in and out of the nucleus?
mRNA (out) Transcription Factors (in) Ribosomal proteins (in)
Transcription Factors
Proteins which activate gene expression and bind to specific promotor regions
In one second
1000 proteins pass through a pore
Ribosomal proteins are transported IN because
rRNA is inside the nucleus
Too complex a process for the cytoplasm
The ribosomal subunits are transported
Out of the nucleus
NLSs
Nuclear localisation signals
NLSs can be located
Anywhere on the protein
NLSs are
Positively charged residues
Only one protein in a complex needs to have a NLSs for
All subunits to be transported
Nuclear import receptors
Recognise NLSs directly or indirectly
‘S’ shaped with a pocket to bind cargo
Karyopherins, Importins
Another name for nuclear import receptors
Nuclear import adaptor proteins
Receptor is binding another protein (an adaptor) that bind the protein
Ran
Small GTPase - molecular switch with two states, active (GTP cound) inactive (GDP bound)
GAPs
GTPase activating proteins
Switch Ran OFF
By increasing rate of GTP hydrolysis
GEFs
Guanine nucleotide exchange factors
Turn Ran ON
By increasing rate of GDP-GTP exchange
Ran-GEF is found in
The nucleus
Ran-GAP is found in
The cytosol
Karyopherins in the cytosol
Do NOT bind Ran-GDP
Therefore cytosolic Karyopherins are free to bind cargo
Karyopherins in the nucleus
DO bind Ran-GTP
Causing a confromational change in the Karyopherin that releases the cargo
Karyopherins that get transported back into the cytosol
Ran-GTP is hydrolysed by GAP and Karyopherin is released to allow more cargo to bind
Ran-GDP is transported
Back into the nucleus via its own nuclear transport receptor and converted back to Ran-GTP by GEF
Nuclear export occurs when
Specific proteins interact with Karyopherins in a Ran-GTP specific manner
Nuclear import and export is regulated by a
Molecular switch
Ran-GTPase
Transmembrane transport into the mitochondria is special because
It has a double membrane
Proteins can be in the outer membrane, the intermembrane space, the matrix, or the inner membrane
Mitochondria in live cells look like
Spaghetti or worms
Interconnected networks, highly dynamic, fusing and dividing all the time
Proteins to be imported into the mitochondria are
Synthesised in the ribosomes, but not folded
Chaperones
Are proteins that bind and block folding
Signals that determine entry into the mitochondria matrix
Form amphiphilic alpha helices with positively charged clusters on one side
Mitochondrial entry signals are recognised by
Protein translocator complexes TOM and TIM
TOM
Translocator outer mitochondrial membrane complex
TIM
Translocator inner mitochondrial membrane complex
TOM transports proteins across the outer mitochondrial membrane by
Binding to the signal sequence and hydrolysing ATP
This causes dissociation of Chaperones
Hsp60 and 70
Family of chaperone proteins in the matrix of mitochondria that fold proteins
TIM transports proteins across the inner mitochondrial membrane by
Binding to the signal sequence and using the inner membrane potential to drive the positively charged residues into the matrix
Signal sequences directed to the mitochondria are
Positively charged
Once the protein is inside the matrix it is immediately bound by
Hsp70 (chaperone protein)
ATP hydrolysis releases
Hsp70 from the polypeptide
Hsp60
Folds the protein in the matrix once Hsp70 has been released
Inner mitochondrial membrane proteins have either:
- A hydrophobic region region following the positively charged signal sequence - this stops TIM23 from translocating the protein through to the matrix
- A second signal sequence facilitating transport back from the matrix to the inter membrane space via the OXA translocator
- Metabolite transporters are pulled through TOM as a loop, and bound by intermembrane chaperones. These guide the transporters to a second TIM complex (TIM22) which inserts them into the membrane
Intermembrane space proteins either
- Have a sequence that stops TIM23 moving the protein into the maxtrix or cytosol (so remains in the space)
- Get transported through OXA and then have the signal cleaved by a specific peptidase
Method to study the mechanism of protein translocation (4)
In vitro radiolabelled translated protein incubated with and without purified organelles:
- Centrifuged proteins that have gone into the organelle will pellet with the organelle, proteins which have not will be in the supernatant
- SDS page analysis will show proteins which have had sequence cleaved (lower MW so will travel faster through the gel)
- Incubate organelles with proteases, proteins that have not been translocated will be degraded
- Genetics: yeast cells have histidine in the cytoplasm, can artifically add a ER targeting sequence to HisDH, so cell will not generate histidine and cells die. Mutating the His gene will allow the mutant cells to live. Mutated protein must be involved in translocation
Chloroplast transport is similar to mitochondrial transport but requires
An additional step
Different topological problems
In chloroplasts
A second hydrophobic sequence is unmasked after the first is cleaved in the stroma
Chloroplasts use
Energy from GTP and ATP to get photosystem proteins across a double membrane
Then a H+ gradient across the thylakoid membrane
