Semester 1 Cell Biology Flashcards
What is the evidence of mitochondria originating from an endosymbiotic event?
- Double membrane
- Presence of cDNA with bacterial origin
- Mitochondrial specific transcription/translation
What does 16kbp human mitochondrial DNA encode for?
- 13 respiratory chain proteins
- rRNA : large and small ribosomal subunits
- tRNA to support translation
From which ancestor did mitochondria derive and what are the key facts about the origination and genes of mitochondria?
- Derived from the ancestor of Rickettsia prowazekii
- Originated from a single event
- Genes found universally in all mitochondria
What are mitochondria?
Organelles responsible for production of energy for the cell
What reactions occur in the mitochondrial matrix?
- TCA Cycle
- Beta-Oxidation of fatty acids
- Urea cycle
- Amino acid biosynthesis
- Mitochondrial protein synthesis
What does the TCA cycle do?
The TCA cycle produces biosynthetic precursors
It provides starting material for:
- Amino acids
- Porphyrins (haem, chlorophyll)
- Purines and Pyrimidines
After the starting material is created, synthesis can occur:
- In the mitochondrial matrix itself for things such as porphyrin
- Following export of the starting material to the cytoplasm such as fatty acid biosynthesis
What is unique about mitochondrial membrane composition?
Inner membrane very high concentrations of protein to accommodate the ETC
Inner membrane has high concentration of cardiolipin
Both inner and outer membrane are poor in sterols, wont find cholesterol
Structure of cardiolipin?
Basically two phospholipids joined together
Headgroup:
- Contains a glycerol that bridges two phosphatidic acids
- Has a double negative charge making it an anionic lipid, which is functional for cytochrome c
Acyl Chains:
- Cardiolipin has four acyl chains per lipid
Volume: The chains occupy a significant volume.
Summarise the evolution and roles of the outer mitochondrial membrane’s interface with the cell
- Initially a passage for energy substrate exchange
- Outer mitochondrial membrane evolved into a communication centre
- Now oversees processes like apoptosis and mitophagy and connects mitochondria to other energy-reliant organelles.
Overview of the Outer Mitochondrial Membrane (OMM)?
- High permeability
- Viewed as analogous to bacterial membrane, however, has unique lipid composition
- Enriched with porin-like proteins
- Also possess helical membrane proteins
- These proteins facilitate metabolite transport across the OMM
What is the most abundant porin-like proteins in the OMM?
Voltage dependant anion channel (VDAC)
Provides a low barrier to exchange of ATP/ADP
Voltage Dependant Anion Channel (VDAC) Structure?
Composed of 25 beta strands arranged in a barrel like structure
Outside of the barrel interfaces with the lipid bilayer
Inside provides a channel through which substrate can pass
Channel is lined with positive charges, providing selectivity for negatively charged molecules
Contact sites between OMM/IMM rich in VDAC
Primary functions of OMM in cellular structure and regulation?
Mechanical Links:
- Connects with other organelles (e.g, ER, endosomes)
- Interacts with the cytoskeleton
Regulation of Cellular Processes:
Apoptosis:
- Involves BH3 family of proteins (e.g., Bax/Bcl2)
Mitophagy:
- OMM’s role in mitochondrial degradation
Where are mitochondria generally found?
Mitochondria tend to be aligned along microtubules
Size and location of mitochondria?
The size and location of mitochondria is dynamic
Why is mitochondrial transport essential in cells, and why do mitochondria need proximity to the nucleus?
- Transport is crucial for cellular function
- Mitochondria require nuclear proteins, necessitating their proximity to the nucleus
With neurones, what are the challenges regarding mitochondrial transport, and how are they typically distributed in terms of mobility?
- Neurons have extended structures, requiring mitochondria at distant sites for functions like Ca2+ regulation and ATP synthesis
- About 70% of mitochondria in neurons are stationary, while 15% are undergoing transport
How do mitochondria move within cells?
- They move along microtubule tracks driven by motor complexes
- Specifically Kinesin for anterograde transport and Dynein for retrograde transport
- Both Kinesin and Dynein share structural homology with a myosin headgroup and act in a similar manner
What is the role of Miro and Milton in mitochondrial transport
- Miro (an integral OMM protein) and Milton (an adaptor protein) link the motor complexes to the mitochondria
Why might mitochondria remain immobile in areas with high levels of Ca2+?
High Ca2+ areas often correspond to metabolically active regions. The binding of Ca2+ to MIRO through its two EF hands could also cause this immobility.
Mechanisms of Mitochondrial Anchoring
- Interaction of OMM-associated myosin with the cell’s actin network.
- Interaction of Synaptophilin with the microtubule.
How can Ca2+ impact the association of the motor complex with microtubules?
Binding of Ca2+ to the EF hands in Miro leads to a conformational change in Miro, causing the dissociation of Kinesin from the microtubule.
How does the PINK1/Parkin pathway influence mitochondrial trafficking?
It results in the ubiquitinylation and degradation of Miro, leading to mitochondrial dissociation. This pathway targets mitochondria with poor electrochemical potentials for autophagy, with Parkin being recruited by accumulated Pink1 on the membrane.
List the four mechanisms that can stop mitochondrial movement.
- Myosin on mitochondria tethering to the axonal actin cytoskeleton
- Syntaphilin anchoring mitochondria through interactions with microtubules
- Ca2+ binding to Miro rearranging the motor domain of kinesin, preventing it from binding to microtubules
- The PINK1/Parkin pathway causing Miro degradation, leading to irreversible dissociation of motors from the mitochondrial surface
What are the key features and functions of the Inner Mitochondrial Membrane?
Site of energy generation
Rich in protein’s involved in respiratory chain
Structure evolved to optimise the role – restricted diffusion, localisation of reactions
Contains transporters to move substrates out into the cell (e.g. ATP, Acetyl-CoA)
What is F1F0-ATP Synthase and what are its primary features and functions?
Overview:
- F1F0-ATP Synthase is a vital enzyme complex found in the inner membranes of mitochondria. It plays a crucial role in producing ATP
Function:
- Converts energy from a proton gradient into ATP.
- This process is often termed oxidative phosphorylation in eukaryotes
Water-soluble head (F1):
- Sits within the mitochondrial matrix
- Directly responsible for the synthesis of ATP
Transmembrane domain (F0):
- Spans the inner mitochondrial (or bacterial) membrane
- Facilitates proton transport back into the matrix (or bacterial interior)
- This transport is coupled to a rotating mechanism, which drives the ATP synthesis in the F1 portion.
What are the main features and functions of the ATP/ADP Shuttle (Adenine Nucleotide Translocator)
Function:
- Facilitates the stoichiometric exchange of ADP and ATP across the mitochondrial inner membrane, ensuring a one-to-one ratio in the exchange process
- Features the “RRRMMM” nucleotide binding motif.
Structure:
- Comprises 2 sets of 6 transmembrane domains (TMD) per dimer
Action Mechanism:
- When ATP binds in the matrix, it triggers a conformational change in the transporter, facilitating the exchange process.
What is MICOS and its primary role?
- Stands for “Mitochondrial Contact Site and Cristae Organizing System.”
- Involved in maintaining mitochondrial architecture
- Drives membrane invagination in mitochondria
- MICOS has two distinct complexes
What is the impact of ATPase dimerisation and Mic60 knockouts on mitochondrial structure?
- ATPase dimerization drives membrane curvature. When abolished (like in su eD), it leads to bridging membranes
- Knocking out Mic60 produces long lamellar-like structures
- Knocking out ATPase results in long lamellar structures linking the sides of mitochondria
How does the Mic60 complex interact and how does cardiolipin play a role?
- Mic60 forms a large, species-dependent complex within MICOS
- The interaction of proteins within this complex depends on the lipid cardiolipin
What are the two primary components of MICOS and their main responsibilities?
Mic10:
- Responsible for membrane sculpting
Mic60:
- Forms contact sites with the outer mitochondrial membrane (OMM)
Describe the significance and interactions of Mic60 in the MICOS complex
- Mic60 forms the core of the MICOS complex
- It interacts with peripheral proteins in the intermembrane space, linking to OMM proteins such as VDAC/TOM and SAM complex
- Forms contact sites between the inner mitochondrial membrane (IMM) and OMM, aligning components for protein import
- Disruption between IMM and OMM interactions disrupts cristae formation
What is the structural and functional significance of Mic10 in the MICOS complex?
- Mic10 has two transmembrane domains (TMD) and a positively charged loop
- It features conserved TMD gly motifs that promote oligomerisation
- Oligomerisation of Mic10 drives membrane curvature
What are the implications of mutations in the MICOS complex?
Mutations in MICOS components are linked to a wide range of diseases
Why do mitochondria have such an elaborate geometry?
- The cristae increase the membrane surface available for enzymes and other vital molecules
- Enables the localisation of reactions: The electron transport chain (ETC) is localised on the cristae
- Ensures efficient substrate transport within the organelle
- The H+ gradient is localised, ensuring efficient energy production
- Restricts membrane protein diffusion, promoting targeted interactions and processes
What is the Chemiosmotic theory?
It is the use of high energy electrons to generate an electrochemical gradient
In mitochondria high energy electrons from oxidation of food are used to generate the electrochemical gradient
In chloroplasts the harvesting of light is used to generate the electrochemical gradient
What is the electrochemical gradient used for?
Used to:
- Power molecular motors that drive ATP biosynthesis (ATPases)
- Drive transport of molecules against their concentration gradients
What is meant by electrochemical gradient in mitochondria?
In mitochondria the electrochemical gradient is generated from protons
The electrochemical gradient has the units kJ mol-1
What are the two components of an electrochemical gradient?
Chemical element (∆pH)
- Difference in concentrations of H+’s across the bilayer
Electrical (∆𝜓)
- Separation of charge across the bilayer
In bioenergetics ∆µH+ is often defined as the proton motive force ∆p with units of millivolts
What are the names of the 5 complexes in the ETC
Complex 1: NADH-ubiquinone oxidoreductase
Complex 2: Succinate-quinone oxidoreductase
Complex 3: Cytochrome bc1 complex
Complex 4: Cytochrome c oxidase
Complex 5: F1F0-ATP Synthase
What is the Electron Transport Chain (ETC) and where is it located?
- A series of protein complexes in the inner mitochondrial membrane
- Each complex is rich in redox centres
- Facilitates the transfer of electrons from donors to acceptors
- Produces a proton gradient for ATP synthesis
- Integral to cellular respiration and energy production
How can we relate ΔEm to ΔG?
The equation:
ΔG= −zFΔE
represents the relationship between the change in Gibbs free energy (ΔG) and the change in electrochemical potential (ΔE) for a redox reaction
‘ΔG’ is the Gibbs free energy change for the reaction. It indicates the amount of energy available to do useful work
‘z’ is the number of electrons transferred in the redox reaction
‘F’ is Faraday’s constant. It represents the electric charge per mole of electrons and has a value of approximately 96,485 C/mol
‘ΔE’ is the difference in electrochemical potential between two half-reactions in a redox process
What redox centres do the mitochondria use?
NAD+ / NADH
- ΔEm of -0.25 V
- Carry 2 electrons
- Also carries 2 protons
FLAVINS
- ΔEm of 0.20 V
- Carry 2 electrons
- Also carries 2 protons
UBIQUINONE
- ΔEm of 0.045 V
- Carry 2 electrons
- Membrane bound large isoprenoid chain
IRON-SULFUR CENTRE
- ΔEm of between -250 to 250 mV, finely tuned by the protein it is in
- Carry 1 electron
CYTOCHROMES
Haem a - ΔEm 385mv
Haem b - ΔEm 0.070mV
Haem c - ΔEm 0.254mV
Describe the function and organisation of the electron transport chain (ETC) in the context of redox centers?
The ETC is an ordered series of redox centers
These centers are organized based on their redox potential
As electrons jump from one centre to the next, ETC complexes couple this to proton transport, creating a proton gradient across the inner mitochondrial membrane.
Explain the function and mechanism of Complex I (NADH/Ubiquinone Oxidoreductase) in the Electron Transport Chain
- Complex I is the largest complex in the ETC and initiates the chain.
It oxidizes NADH, transferring the electrons to ubiquinone (CoQ)
THE MECHANISM INVOLVES:
- Binding of NADH, leading to the transfer of two electrons to FMN (Flavin mononucleotide), converting it to FMNH₂
- These electrons are then sequentially transferred through a series of iron-sulfur clusters
- Eventually, the electrons reduce ubiquinone (CoQ) to ubiquinol (CoQH₂)
For every two electrons transferred from NADH to CoQ, four protons are pumped across the inner mitochondrial membrane, contributing to the proton gradient.
How do electrons transfer between redox centres?
Separation between redox centres is large >10 angstroms
Electrons use quantum tunneling to move between redox centres
Which subunits form the ubiquinone binding site in Complex I, and what is the nature of this site?
The ubiquinone (CoQ or UQ) binding site is formed by the Nqo4, 6, 7, and 8 subunits
The site is hydrophilic in nature, suggesting it plays a role in guiding the ubiquinone headgroup
The UQ binding site is not located near the membrane. Instead, it has a binding pocket both for the UQ head and its isoprenoid chain
Why is the ubiquinone binding site distally placed from the membrane in Complex I, and what are the implications of its placement?
The binding site is positioned away from the membrane due to potential energetics concerns: a 2- charge within the bilayer would be highly unfavorable
The positioning ensures that if the protein controls quinone protonation, the charge can drive conformational changes. Once these changes are completed, the quinone can then be protonated and released
Protons involved in this process come from Tyr (Tyrosine) and His (Histidine) residues.
Describe the role of the Nqo12, Nqo13, and Nqo14 subunits in Complex I
Nqo12, Nqo13, and Nqo14 function as antiporters in Complex I
They possess 14 conserved transmembrane helices (TMH), with 10 of them in the functional core region
Their structure can be visualized as 2 sets of 5 TMH each, related by a 2-fold screw axis symmetry
They provide the proton conduction pathway, with each subunit contributing a half-channel, which is lined with polar residues and filled with water
A key lysine (Lys) residue acts as a gate, with its pKa modulated by a nearby glutamate (Glu) residue. This arrangement might be a historical relic, hinting at the evolutionary origin of the antiporter
Characterize the Nqo8 subunit in Complex I and its distinctive features
Nqo8 is considered the fourth antiporter of Complex I
It is notably charged for an integral membrane protein
Its fifth transmembrane helix (Tm5) is discontinuous, and Glu residues within it are believed to be involved in proton transport
Nqo8 forms the E channel in Complex I
What likely provides the energy for conformational change in Complex I?
The reduction of N2 to UQ (ubiquinone) in Complex I is the probable source of energy that drives conformational changes
This leads to modifications in the E channel (Nqo8) which are then propagated throughout the other antiporters
How might the ubiquinone dianion (UQ2-) influence conformational changes in Complex I?
It’s believed that UQ2- might interact with a center composed of glutamate (Glu) or aspartate (Asp) residues, initiating significant conformational changes in the protein structure
How do the antiporters in Complex I function in relation to each other?
The antiporters in Complex I operate cooperatively, pushing one another into alternate conformations to ensure efficient proton transport
What role do the transverse helices play in Complex I?
Couple all the of the transport proteins and couple their motions
Transverse helices are vital for stabilizing the entire Complex I structure
They also coordinate the conformational changes essential for its function.
Describe the initial interaction of ubiquinone with Complex III and the first electron transfer
There exists a pool of ubiquinone molecules
Ubiquinone binds on the P face of the mitochondria
UQH₂ donates one electron to the 2FeS2 centre which is located in Rieske proteins
This electron can then be transferred to cytochrome c
As a result, Complex III now houses a ubiquinone radical
Detail the subsequent electron transfer events and proton movements in Complex III
The radical in Complex III transfers its electron to another ubiquinone molecule on the opposite side of the lipid bilayer
This results in the creation of a new ubiquinone radical
During the oxidation of UQH₂, two protons are released to the P side of the mitochondria
As the binding site clears up, another UQH₂ molecule binds to it
Similarly, one electron from this UQH₂ is passed to the 2Fe2S centre (located in Rieske proteins), which then transfers to cytochrome c, releasing two more protons to the P side
Explain the final steps of the Q-Cycle in Complex III
The lingering electron in Complex III binds to the previously formed ubiquinone radical, producing UQH₂ on the N side of the lipid bilayer
This process requires the uptake of two protons from the N side of the mitochondria
The newly generated UQH₂ then becomes part of the circulating ubiquinone-ubiquinol pool
The result is the movement of 2 protons from the N side to the P side of the mitochondria
Why do electrons take different paths in Complex III, and what role does the Rieske protein play?
Electrons from ubiquinol (UQH₂) take different paths to ensure efficient proton pumping and electron transfer within the Q-cycle of Complex III
The Rieske protein contains a 2Fe-2S iron-sulfur cluster, which can change its position relative to other components in the complex
Upon UQH₂ binding and electron uptake, the Rieske protein undergoes a conformational change
This repositioning ensures that one electron is transferred directly to cytochrome c via the cytochrome c1 subunit
Meanwhile, the other electron is directed through the b-type cytochromes (bL and bH) to reduce a ubiquinone molecule at the QN site
The distinct paths help create the conditions necessary for the generation and movement of protons across the membrane, contributing to the proton gradient used for ATP synthesis
What are the fundamental characteristics and role of cytochrome c?
Cytochrome c is a small protein weighing 12.5 kDa
It contains a bound haem c group.
Its primary function is shuttling electrons between Complex III and Complex IV
Additionally, the release of cytochrome c following the disruption of the outer mitochondrial membrane (OMM) is a key step initiating apoptosis
Describe the surface properties of cytochrome c and its significance.
Cytochrome c’s surface is rich in positive charge
A small pocket provides access to the haem
This positive charge, especially around the pocket, is thought to mediate electron transfer interactions via charge/charge interactions with other complexes
How does cytochrome c interact with Complex III for efficient electron transfer?
The distance between cytochrome c and the haem of Complex III is minimal, about 9Å
This close proximity promotes rapid electron transfer
The positive charge on cytochrome c also serves as an anchor to the membrane surface, ensuring efficient interaction
How does the charge of cytochrome c influence its behavior in the IMM?
The IMM is rich in cardiolipin (CL), an anionic lipid
The binding of the positively charged cytochrome c to this negatively charged bilayer ensures diffusion within the membrane’s plane
This arrangement promotes rapid electron transit between the complexes, facilitating efficient electron transport
Describe the interaction mechanism of cytochrome c with Complex IV
Cytochrome c binds to Complex IV via weak electrostatic interactions
This weak binding favors the rapid exchange of cytochrome c, optimizing electron transfer dynamics
Detail the electron transfer process involving the CuA centre in Complex IV
Electrons from cytochrome c are first transferred to a CuA centre
Within this centre, two Cu ions are ligated by a cysteine and a histidine from SU-II
This CuA centre undergoes a one-electron reduction
Describe the electron flow after the CuA centre within Complex IV
Post CuA centre reduction, the electron tunnels to cytochrome a with a timeframe of 11 microseconds
It then transfers to the CU-cytochrome a3 complex almost instantaneously, in nanoseconds
How does proton transfer occur within Complex IV?
Proton transfer is mediated by water-filled channels lined with polar amino acids
One primary channel is the “D channel” which runs from Asp D91 up to E242, positioned near the reaction centre
This D channel is speculated to be responsible for pumping 4 protons and delivering 2 protons for the reduction of O2. There’s also mention of a potential “H channel” based on some experimental evidence
What is the significance of the K channel in Complex IV?
The K channel, marked by K319, terminates near a tyrosine that’s crosslinked to the farnesyl group of haem
Provide a summary of the oxygen reduction process in Complex IV
Oxygen reduction in Complex IV proceeds in stages:
Formation of Oxy Species:
- Fully reduced binuclear center binds oxygen at cytochrome a₃
Formation of P Species:
- The oxygen bond breaks, with oxygen atoms binding to cytochrome a₃ and CuB, facilitated by electron transfers from cytochrome a₃, CuB, and a conserved tyrosine
Formation of F Species:
- Electron and proton deliveries restore the tyrosine
Hydroxyl Formation:
- Additional electron reduces cytochrome a₃, converting the bound oxygen to hydroxyl
Restoration & Water Formation:
- Further electron and proton deliveries restore original redox states, releasing two water molecules
Describe the initial binding of oxygen in the fully reduced binuclear center of Complex IV.
The binuclear center is in its fully reduced state
Oxygen binds specifically to cytochrome a₃, leading to the formation of the “oxy species.”
How does the formation of the P species occur, and what changes in the oxidation states of the components?
The oxygen-oxygen bond breaks: one oxygen atom stays bound to cytochrome a₃, while the other binds to CuB
Four electrons are transferred to the oxygen atoms:
- Two from cytochrome a₃, turning it to a 4+ ferryl state
- One from CuB, changing it to a 2+ state
- One from the conserved tyrosine, converting it to a neutral free radical after donating a proton to the oxygen atom on CuB
How is the F species formed in Complex IV?
An electron is delivered from cytochrome c via CuB and cytochrome a₃
Concurrently, a proton from the N-phase (matrix side) restores the tyrosine to its non-radical state, completing the formation of the F species
Explain the changes occurring in cytochrome a₃ during the fourth stage of oxygen reduction
An additional electron reduces cytochrome a₃ to its 3+ state
The bound oxygen on cytochrome a₃ acquires a proton, transforming it into a hydroxyl group
What is the final outcome in the oxygen reduction process of Complex IV?
A further delivery of two electrons and two protons restores both cytochrome a₃ and CuB to their original redox states
This results in the formation and release of two water molecules from the complex
Structure of F1F0 ATPase?
Can be broken down into two main regions:
F1 Head:
- Site of ATP synthesis
- Catalytic domain
Stalk domain (couples F1 and F0)
F0 Complex:
- Buried in membrane of mitochondria
- Motor unit
What are the subunits of F1F0 ATPase?
F0 motor structure?
Composed of two types of subunit:
a:
- Stator, provide half channels for translocation of proton across the bilayer.
c:
- Rotor, 8 copies in mitochondria
Role:
- Couple movement of protons across the bilayer to the rotation of the central stalk.
Negatively charged glutamate halfway through the bilayer
Glutamate ‘picks up protons’
What are the Key Structural Components and their Initial Proton Interactions in F₁F₀-ATPase?
a subunit: Contains two half-channels for proton passage
c-motor ring: Integral for proton transport and rotational mechanism
Key residues:
- R239 (argenine) in a subunit: Acts as a blockage, dictating proton transit
- Conserved glutamate residue is a target for proton entry, and when deprotonated, influenced by Δp (Proton Motive Force)
- Conserved glycine residue: Plays a role in the proton acceptance mechanism within the c-ring
How Does the Proton Motive Force Influence c-ring Rotation?
Δp (Proton Motive Force):
- Primary driving force for the system’s motion
- Applies force on the deprotonated Glu situated in the lipid bilayer
Resultant Action:
- The influence of Δp on Glu generates enough force to drive the rotation of the c-subunit ring
How is the Proton Released and What Concludes the ATP Synthesis Process?
- Proton transfers to the Glu residue on the c-ring, allowing rotation
- As the c-ring rotates, the protonated Glu confronts R239 (argenine)
- This interaction compels Glu to release the proton into the second half-channel
- The proton then ventures out, either into the mitochondrial matrix or the chloroplast stroma, completing the ATP synthesis process
Why Does the c-ring of F₁F₀-ATPase Rotate?
Proton Motive Force (Δp):
- A combination of a proton concentration gradient and an electric potential difference
- Drives protons across a membrane
Mechanism:
Proton Entry:
- Through a half-channel in the a subunit, targeting a conserved glutamate on the c-ring
Charge Neutralisation:
- Protonation of glutamate induces a charge change, prompting movement
Rotation Initiated:
- Altered interactions due to charge change cause c-ring rotation
Proton Exit:
- Protonated glutamate aligns with the second half-channel and, upon encountering the charged residue R239 (arginine), releases the proton.
Continued Rotation:
- Consistent proton inflow perpetuates c-ring rotation.
How does stalk rotation affect interaction with F1
The stalk is asymmetrical, depending on the stage of its rotation, the offset inside of the F1 will be different
For example:
4 protons = 100 degrees movement
4.4 protons = 112 degrees movement
5.6 protons = 156 degrees movement
This results in 3 different states of the stalk
This causes perturbation in the F1 domain
F1 domain structure?
3 alpha/beta domains that form 3 alpha/beta dimers
Each alpha/beta dimer will have:
- 1 ATP bound
- 1 ADP + Pi
- 1 Empty
What does the stalk (gamma-subunit) perturbation result in, in the F1 unit?
As it rotates and perturbs the F1 subunit, different states of the stalk causes a perturbation in the nucleotide binding sites
This perturbation of the nucleotide binding sites drives ATP synthesis
How does rotation of the gamma-subunit (stalk) drive atp synthesis?
Rotation of g-subunit:
- Convert ATP site to open site, allowing release of ATP
- Converts ADP/Pi into a tight binding site
This allows:
- The binding of ADP/Pi to the new empty site.
- Conversion of ADP/Pi into ATP
Events are cooperative, coordinated action between 6 subunits
Give an overview of the Endomembrane System
Definition:
- A coordinated system within eukaryotic cells that divides the cell into distinct membrane-bound compartments
Functions:
- Regulates translation, modification, and trafficking of proteins
- Modulates signal transduction pathways
Components:
- Nuclear envelope
- Endoplasmic reticulum
- Golgi apparatus
- Plasma membrane
Describe vesicle transport and give its additional components
Vesicle Transport:
- Critical process in maintaining and regulating the functions of the endomembrane system
Role:
- Ensures proteins and lipids are correctly sent to their intended destinations and can aid in intracellular digestion and waste removal
Key Vesicular Structures:
- Secretory vesicles
- Endosomes
- Lysosomes
- Autophagosomes
Vesicle transport basic overview
Vesicles bud off and fuse with different compartments
Carry ‘cargo’ – membrane associated and soluble molecules
Each vesicle must be selective for certain cargo and fuse with appropriate target membrane
What are the two pathways for vesicle flow?
Secretory pathway:
- Flow of membrane bound and soluble proteins destined for certain organelles or extracellular space flow from ER to Golgi, to plasma membrane via secretory vesicles
Endocytic pathway:
- Plasma membrane capture of extracellular components and internalisation of membrane proteins into vesicles that result in recycling of receptors or degradation of contents in the lysosome
How is vesicle transport visualised?
Visualising protein transport through the secretory pathway
A viral membrane glycoprotein VSV-G was labelled with GFP
It is a temperature sensitive reporter that can be synchronously released from ER and allowed to transport to Golgi and onwards to plasma membrane
What are some requirements for vesicle transport?
Identification of specific cargo
Sorting of vesicles and associated cargo
Transport
- Cytoskeletal motor proteins
Transfer of vesicular material
- Fission
- Tethering
- Fusion
What are vesicle coat proteins?
The transport vesicles usually have coat proteins that:
- Provides shape to membranes to “curve” and bud
- Determine the size and shape of the vesicle
- Concentrate the protein in the vesicle
- Provide selectivity for the “cargo”
- Determine the vesicle’s destination
What are some types of vesicle coating proteins and where are they found?
Clathrin coated vesicles
- Trans-Golgi network (TGN) to endosomes
- Plasma membrane (via endocytosis)
COPI coated vesicles
- Golgi complex to the ER (retrieval)
COPII coated vesicles
-ER to Golgi
What are some ways proteins associate with the lipid membrane?
Embedded in lipid membrane
Partially imbedded in membrane
- Hydrophobic region that allows them to embed themselves in the membrane
Proteins that are embedded via a covalently attached lipid group (fatty acid or prenyl group)
Proteins that undergo non covalent interactions with other membrane bound proteins (peripheral membrane protein)
clathrin coated vesicles structure and use
Transport material from plasma membrane and between endosomes and Golgi apparatus
Clathrin subunits are made up of 3 large (heavy chain) and 3 small polypeptides (light chain) that assemble in “triskelions” at the trans-Golgi network (TGN) or at plasma membrane
Clathrin forms an outer protein lattice
What is endocytosis?
Engulfment of extracellular molecules occurring at the plasma membrane
Regulates
- Receptor signalling
- Receptor turnover
- Nutrient uptake
- Polarity
- Cell migration
- Neurotransmission
Various types:
+ Receptor-mediated endocytosis
- Clathrin-dependent
- Caveolin-dependent (lipid rafts, sphingolipids, GPI
anchored proteins)
- Clathrin- and Caveolin- independent
+ Phagocytosis
+ Pinocytosis
How does the clathrin coat form?
During endocytosis at plasma membrane
Recruitment of AP2 adaptor protein complex is required for:
- Clathrin recruitment
- Coat assembly (formation of clathrin-coated pits),
- Eventual budding
AP2 adaptor protein binding to specific phospholipids results in conformational change that allows binding to cargo receptors on cell surface, triggers membrane curvature
AP-2 Adaptor Protein Complex Structure and what does it do?
AP-2 adaptor protein complex:
- Heterotetrameric
Multi-subunit:
- α-adaptin
- β2-adaptin
- σ2-chain
- µ2-chain
AP-2 on clathrin coated vesicles originates from the plasma membrane (endocytosis)
AP-1 adaptor complex originates from (Golgi)
Recognises specific peptide motifs on the cargo receptor (endocytosis signals)
Interacts with:
- Plasma membrane lipids
- Cargo
- Clathrin
AP adaptor activation and clathrin coat formation?
AP-2 adaptor complex is formed in the cytoplasm, but kept in locked/inactive position
Clathrin binding site is buried in AP-2 complex in ‘locked’, soluble state (not bound to membrane)
Binding to PIP2 on membrane exposes clathrin binding motif in beta2-adaptin leading to a transition to the AP-2 ‘open’ conformation
µ2-subunit at same time interacts with cargo which further stabilises AP-2 complex ‘open’ conformation and the dwell time of AP-2 at plasma membrane – thus facilitating clathrin coat assembly
Assembly of clathrin coat?
- Protein “cargo” binds to a membrane bound receptor protein (e.g. mannose-6-P receptor on golgi)
- The receptors only selectively recruit the correct cargo for the vesicle
- The receptor also bind adaptor proteins (e.g. AP-1 complex), which in turn binds to the triskelion clathrin
- Many “cargo-receptor-adaptin-clathrin” complexes form in a clathrin-coated vesicle
- Vesicle formation and budding is assisted by dynamin (requires GTP)
- The clathrin coat dissociates immediately & components are recycled – leaving behind an uncoated vesicle that is transported to its destination
How is membrane fission facilitated by dynamin?
Dynamin dimers oligomerises and forms a helical ring around the neck of the bud, recruits other proteins, and tethers itself to the membrane through lipid binding domains
Dynamin constricts in the presence of GTP
GTP hydrolysis of dynamin results in the lengthwise extension of helix, and fission of membrane
How can dynamin function be visualised and understood experimentally?
Use of temperature sensitive mutants to understand dynamin function in vesicle scission
Ts mutations in dynamin (e.g. Drosophila “Shibire”) halt vesicle fission and allow visualisation of arrested buds
Results in immediate paralysis of the flies – but is reversible upon return to permissive temperature
Initiation of COPII Vesicle Formation
Sec12:
- ER membrane-localised guanine nucleotide exchange factor (GEF)
Sar1 Activation:
- Sec12 catalyses the exchange of GDP for GTP on Sar1
- Activation leads to Sar1’s association with the membrane.
COPII Vesicle Coat Formation and Cargo Selection
Sec23 & Sec24 (Inner coat):
- Form complex binding to Sar1-GTP
- Sec24: Binds ER-export signals on cargo proteins
- Sec23: Aids in coat assembly and Sar1 GTPase-activating activity
Sec13 & Sec31 (Outer coat):
- Recruited by sec23 and sec24
- Assemble into lattice structure over inner coat for curvature and budding
Cargo Selection:
- Cargo proteins with ER-export signals incorporated into vesicle via Sec24.
COP I Retrograde Transport Method?
COP I coated vesicles retrieve proteins from Golgi back to the ER
ER proteins have KDEL (Lys-Asp-Glu-Leu) at C-terminus which is recognised by a KDEL receptor in cis-Golgi, and retrieved by interaction with COP I
As you go from ER to Golgi, becomes more acidic
- As pH is lowered, affinity for KDEL receptor is higher
COP I structure?
Consists of 7 core subunits
Organised as a cytoplasmic heptamer called coatomer and is recruited en bloc to the membrane
Coat is organised first in cytosol and then recruited at membrane sites to form coated vesicle
ARF1 GTPase is required for coatomer recruitment, which is recruited and activated by Golgi-localised GEF proteins
These GEFs replace GDP with GTP to activate ARF1
What is the importance of GTPases in coated vesicles?
Each type of coated vesicle has an associated GTPase
- Sar1 in COPII vesicles, ARF in COPI and clathrin-coated vesicles
GTP loaded ARF or Sar1 binds to effector proteins which facilitates coat assembly
In the presence of non-hydrolysable GTP analogues, all types of coated vesicles accumulate
This is because GTP hydrolysis is required for coat disassembly
Also, dynamin is a GTPase so budding cannot be completed
Numerous GTPases are involved in vesicle transport (Rab family of GTPases)
What is the Ras family of small GTPases?
They are small guanosine triphosphatases (GTPases)
- Contains over 150 human members
Divided into 5 major branches based on sequence
- Ras
- Rho
- Rab
- Ran
- Arf
Binary molecular switches that share common biochemical mechanism
Function as monomeric G proteins – GDP/GTP-regulated molecular switches
Post-translational modifications control sub-cellular localisation and interaction with the proteins that act as regulators and effectors.
What is the Rab GTPase family?
Largest branch of Ras family (Contains 61 members)
Regulates intracellular transport of vesicles and transport of proteins between organelles of the endoctyic and biosynthetic pathway
Does this through interactions with effector proteins – facilitate vesicle formation, budding, transport, and vesicle fusion at acceptor site
The sub-cellular localisation and specificity for different intracellular compartments of each Rab is dependent on post-translational lipid modifications (prenylation) and effector interactions
How is Rab GTPase activated?
GDI keeps Rab inactive in cytosol, sequestered away from membrane
GDF – GDI displacement factor:
- Displaces GDI from GDP bound form of Rab, thus allowing membrane anchor with its hydrophobic prenyl group
GEF mediated GDP to GTP exchange triggers a conformational change in the Switch 1 and 2 regions of Rab allowing interactions with effector proteins
Rab5 activation example
DOES NOT NEED TO BE MEMORISED, OVERALL UNDERSTANDING OF HOW Rab GTPases ARE RECRUITED AND ACTIVATED IS REQUIRED HOWEVER
A Rab5-GEF binds Rab5 and activates it by exchanging GDP for GTP
GDI is lost and GTP binding induces conformational change that exposes prenyl lipid group anchoring it to the membrane
Active Rab5 can now bind effector proteins (e.g. PI3kinase)
This changes the lipid composition which works collectively with Rab5 to recruit effector proteins (e.g. vesicle tethering proteins, signalling proteins, scaffold proteins)
How does the Rab cascade and vescile identity occur?
As vesicles traffic along the pathway, they change composition and mature – acquiring different components required for transport/function (e.g. SNAREs)
Activation of a RabA-GEF to membrane, locally activates RabA
RabA then activates effector proteins, one of which is a RabB-GEF
Activation of RabB by the RabB-GEF, activates RabB effector proteins, one of which is a RabA-GAP
This RabA-GAP inactivates RabA
Thus, this cycle replaces a RabA domain on a membrane with RabB
This sequence will continue via recruitment of the next GEF by RabB
Vesicle mechanism of tethering target membrane
Once made and budded off, vesicles have to be uncoated to expose the v-SNARE protein
Each v-SNARE protein on the surface of the vesicle has a corresponding t-SNARE protein on the target membrane
GTP-bound Rab protein binds a tethering protein (effector protein)
- e.g. multi-subunit HOPS complex
Facilitates SNARE assembly and vesicle fusion
How do transport vesicles identify their target membrane?
SNARE proteins are used (around 35 in mammals)
Exist in pairs in the membranes:
v-SNAREs on surface of vesicles
t-SNAREs on membrane of target
v- and t-SNAREs have helices that interact with one another and dock the vesicle to the target membrane
The interaction is initiated by a vesicle-specific Rab GTPase
How does vesicle docking occur?
Rab-GTP protein on vesicle surface binds to specific Rab-effector in target membrane
This brings v-SNAREs and t-SNAREs into close proximity -allowing docking
alpha-helices of v-SNARE and t- SNARE form coiled-coils (trans-SNARE complex)
exerts inward force that brings the two membranes close together
How does membrane fusion occur between vescile and membrane?
Mechanism of membrane fusion is unknown, but may be opposite to dynamin model:
- Lipid bilayers fuse by flowing into each other after being forced into close proximity
- A complex of two proteins (NSF and a-SNAP) binds to the “empty” SNARE complexes (cis-SNARE complex)
- ATP hydrolysis (catalysed by NSF) causes disassembly of the SNARE complexes and recycling
What is the mechanism of coordinated synaptic membrane fusion and neurotransmitter release?
Synaptic vesicles dock at presynaptic plasma membrane, with complexin keeping the trans-SNARE complex in a primed position
Calcium induces a conformational change in the complex allowing coordinated vesicle fusion with plasma membrane leading to neurotransmitter release
STUDY PHOTO
Overview of transport of vesicles from endocytosis to a lysosome
Endocytic routes originate from plasma membrane
Maturation of vesicles is required – acquisition of proteins to facilitate sorting, transfer, transport, and vesicle fusion
Eventual fusion of transport vesicles with the lysosome for degradation and recycling of contents
Similarity between golgi derived lysosomal enzyme traffic and plasma membrane derived endocytic vesicle traffic to lysosomes
Golgi derived lysosomal enzymes traffic through subcompartments before making way to lysosome
Plasma membrane derived endocytic vesicles traffic through intermediate compartments before fusing with lysosome
Both require cargo transfer and machinery to become competent for fusion with lysosome
Plasma membrane derived endocytic vesicle transport
Cargo traffics through intermediate compartments along the endocytic pathway
Route from plasma membrane:
- Early endosome
- Late endosome/multivesicular body
- Lysosome
If cargo (i.e. receptor) is recycled
- Early endosome
- Recycling endosome
- Plasma membrane
What are multivesicular bodies?
One of these intermediate compartments used in vesicle traffic is a Multivesicular Body (MVB)
MVBs have a lower pH and contain intraluminal vesicles
Provides mechanism to shield receptors from acidic cytosol, thereby turning off potential signal transduction
Requires a specialised set of machinery for intraluminal vesicle formation
Why do we need multivesicular bodies?
Receptors at plasma membrane get activated by ligand-binding (e.g. Growth factors)
This leads to activation of a signalling cascade that results in changes in cell function (e.g. proliferation, changes in cell shape or gene expression)
These signals need to be controlled
One method of control is the internalisation and inactivation of the ligand activated receptor
Why do we need to internalise receptors?
Following coated vesicle formation, coat disassembles and vesicle is transported to target
However, signalling domain in receptor is still exposed to cytosol allowing it to continue activating downstream events
As ligand-receptor is internalised it gets sequestered and transported to lysosome
This results in degradation of receptor and inactivation of signalling cascade
What is receptor ubiquilation?
Activated signalling receptors at plasma membrane get internalised and trafficked via an endosome
These activated receptors get ubiquitylated
Ubiquitylation acts as signal for receptor sequestration within an intraluminal vesicle
Once a multi-vesicular body forms, it fuses with the degradative lysosome to breakdown and recycle components into building blocks (lipids, amino acids)
What are Endosomal sorting complexes required for transport complex (ESCRT) ?
ESCRT (endosomal sorting complexes required for transport) complex is required for intraluminal vesicle formation
ESCRT-0 contains ubiquitin binding domain which interacts with ubiquitylated receptor cargo
ESCRT-0 also contains binding domain for interaction with PI3P rich phospholipid on endosomal membrane
Multiple subsequent ESCRT proteins help shape the membrane to form an invagination and eventual budded intraluminal vesicle
Variation in membrane phospholipid
Similarly to how there is variation in Rab’s there is also variation in lipid content in membrane phospholipids
Early endosomes acquire the phosphoinositide species PI(3)P in their membrane, which acts as binding site for effector proteins
Different phospholipids recruit different features to aid in vescile formation and transportation
How does the ESCTR complex drive intraluminal vesicle formation?
Cargo (e.g. membrane receptor) ubiquitylation and endocytosis initiates the process
ESCRT-0, -I, -II complexes bind to ubiquitylated cargo and the membrane phospholipid PI3P
ESCRT-III complex and Vps4 are necessary for membrane budding and scission
Hrs is an ESCRT-0 protein that interacts with ubiquitin on cargo
VPS4 (Vacuolar protein sorting-associated protein 4) is an ATPase that hydrolyses ATP to disassemble ESCRT complex allowing intraluminal vesicle to form
How does viral shedding use ESCRT?
Virions bud off from plasma membrane surface using the ESCRT machinery
Requires similar membrane bending to intraluminal vesicle formation
ESCRT machinery location on inside of cell, so pushes budding vescile outwards with virus to infect neighbouring cells
What shape are budding vesicles formed by viral shedding and intraluminal vescile formation?
Shape of budding membranes are similar when comparing viral shedding from plasma membrane and intraluminal vesicle formation in multivesicular bodies
How do components that are soluble within the cytoplasm (not membrane bound) get targeted to the lysosome?
Via Autophagy
What is autophagy?
Autophagy is a cytosolic degradation pathway
Requires autophagosome – double membrane bound organelle
Can degrade misfolded proteins, damaged organelles, or invading pathogens that escape out of phagosome into cytosol
Also can capture bulk cytosol to harvest energy and amino acids during times of starvation
Autophagy capture mechanism?
Requires capture of cytosol or selected target and formation of double membrane autophagosome around cargo
These lipid membranes that form the autophagosome are derived from other intracellular membranes (e.g. ER, plasma membrane, Golgi)
This autophagosome gets sealed and then is transported to the lysosome
Once fused with lysosome contents get broken down into building blocks – lipids, amino acids
What are the autophagy machinaries?
Can select cargoes to be captured by autophagosome (1)
Can also non-selectively capture cytosol during starvation
Both these cases require similar autophagy machinery:
- Atg8
- Atg5-Atg12-Atg16 complex
Atg8 (also known as LC3) is a membrane protein that decorates inner and outer leaflets of autophagosome
Autophagosome maturation and lysosome fusion
Following closure of the autophagosome, there are fusion events with endosomes and MVBs to form an amphisome
This also results in a gradual reduction in internal pH and acquisition of machinery to facilitate fusion with the lysosome (e.g. SNARE components)
Leading to eventual fusion with the lysosome to form an autolysosome, resulting in proteolytic degradation of components
Autophagy summary
During nutrient starvation leading to low ATP levels or low amino acids, autophagy is activated resulting in the removal of bulk cytosol for harvesting of amino acids required for protein synthesis and energy production (non-selective)
Can also be a selective process, targeting specific cargoes such as misfolded proteins or damaged organelles
Here those damaged cargoes are decorated with polyubiquitin chains, which are recognised by specific autophagy receptors
These autophagy receptors have binding sites for polyubiquitin and the membrane associated Atg8/LC3 protein
Therefore, these autophagy receptors help identify cargo to be degraded and direct the recruitment of autophagosome membrane
How does non-selective vs selective autophagy work?
Autophagy receptors can interact with polyubiquitin on cargo and also with Atg8/LC3 on autophagosome membrane to connect the two together
Can either generate membrane around random cytosol cargo
or
Polyubiquitin tags interact with autophagy receptors to bind the cargo to the Atg8 on autophagosome membranes
Actin cytoskeleton
Are polarised, with (+) and (-) end
Composed of G-actin subunits that assemble into F-actin filament
ATP-G-actin gets loaded on filament and undergoes hydrolysis before release from filament
Undergo actin treadmilling
At physiological concentrations – G-actin gets preferentially added to (+) end, while being preferentially disassembled from (-) end
Actin and vesicle transport
Proteins associated with endocytic vesicles and the clathrin coat also recruit actin nucleation-promoting factors
WASP is one of these nucleation promoting factors that activates Arp2/3 complex
Arp2/3 promotes actin polymerisation which drives internalised vesicles away from the plasma membrane
What is WASP and how does it cause Arp2/3 activation
Wiskott Aldrich Syndrome protein (WASP) is an actin nucleation promoting factor
WASP is held inactive (folds back on itself) in cytosol through intramolecular interaction that masks WCA domain
Following interaction with active GTPase (Cdc42-GTP) through rho binding domain (RBD) motif, intramolecular interaction is relieved and W domain is exposed to bind actin and the A domain activates Arp2/3
What happens when WASP binds and activates Arp2/3
WASP binding to Arp2/3 results in conformational change in complex and allows Arp2/3 to bind to a pre-existing actin filament
Actin subunit gets brought in by W domain of WASP and together binds to Arp2/3 to initiate actin nucleation
Nucleation at (+) end occurs and filament extends
Angle between old and new filament is 70°
How does listeria infect cells?
Listeria uses similar arp2/3 methods for motility
Listeria monocytogenes – a food-borne pathogenic bacteria causes gastroenteritis
It enters the cell, divides, and hijacks the cell motility machinery for its own gain
To move from one cell to the other, it moves inside the cell by polymerising actin into a comet tail and pushes its way through the plasma membrane to get to the adjacent cell
Listeria infection and motility mechanism
Arp2/3 complex first discovered studying Listeria movement
Listeria has on its surface a protein called ActA which is a nucleation-promoting factor (like WASP)
ActA activates Arp2/3, which promotes actin filament assembly
Filaments grow at (+) end and get capped by CapZ
Actin is recycled through function of disassembly factor cofilin, which enhances depolymerisation at (-) end
How do vesicles move with the cytoskeleton?
Newly formed vesicles associate with the actin cytoskeleton through adaptor proteins
Actin motor proteins called myosins either tether vesicles to actin cytoskeleton or transport vesicles along the actin cytoskeleton
This facilitates cargo delivery and fusion events
Myosin motor proteins structure
Many different classes of myosins
Each myosin is composed of an actin binding head domain and lever arm neck domain
They may also contain a cargo binding tail domain that interacts with membrane lipids or adaptor proteins
All motor head domains convert ATP hydrolysis into mechanical movement
In case of class I and V, may function as monomer or dimer to tether or transport cargo along actin filament
What is myosin V’s function in organelle transport
Functions as a dimeric motor protein
Moves towards (+) end of actin filament
Has a step size of 30-40 nm, which is ATP dependent
In budding yeast, myoV is essential to transport mitochondria to newly formed bud
What are the two types of microtubule motor protein and what do they do?
2 types of microtubule motor proteins:
- Dynein – move towards (-) end
- Kinesins – move towards (+) end
Transport vesicles or organelles
Involved in long range and fast transport (compared to short range and slower transport of myosins)
Movement from ER to Golgi
ER where membrane and soluble, secreted cargo is translated and trafficked towards Golgi
Intermediate compartment between ER and Golgi, called vesicular tubular cluster or ER-Golgi intermediate compartment (ERGIC)
COPII vesicles lose their coats and fuse to form the vestibular tubular cluster
This organelle serves as a sorting station for cargo between ER and Golgi
How do microtubules maintain ER and Golgi structure
Microtubule motor proteins are essential to maintain the structure and organisation of the ERGIC and the Golgi
Dynein dependent and kinesin dependent transport of vesicles
Dynein motors are essential for Golgi positioning – aligns in perinuclear region and along axis of cell polarity
What are golgins?
Golgins are large proteins (over 30 genes), with coiled-coil domains adopting a rod-like shape
Golgins involved in transport and vesicle tethering around regions of the Golgi
Act as Rab effector proteins
Golgins interact directly with microtubules, with microtubule associated proteins or microtubule motors, such as dynein
Contribute to Golgi positioning and morphology
How does microtubule mediated transport along endocytic route occur
Kinesin- and dynein-dependent transport essential along endocytic route to transport vesicular cargo between various compartments
Lysosomes are positioned in perinuclear regions - loss of dynein leads to a dispersal of lysosomes throughout the cytoplasm
Dynein structure
One gene encoding cytoplasmic dynein heavy chain
Cytoplasmic dynein complex contains a pair of identical heavy chains (homodimer)
Dynein heavy chain has an ATP-dependent motor (head), Microtubule binding stalk region, and N-terminal stem that binds cargo or adaptors
N-terminal stem interacts with intermediate and light chain proteins
Dynein motor requires a complex protein assembly
Dynein-mediated movement mechanism
Each motor head domain contains a hexameric AAA ring that has stalk, buttress, and linker regions protruding from AAA ring
Conformational change of head relative to stem, leading to movement of stalk domain, triggers the power stroke that results in movement
Stalk region interacts with microtubules and the ATP dependent motor domain performs the work
Dynein in presence of ATP has low affinity for microtubules
ATP hydrolysis by AAA domains in motor head leads to microtubule binding
Release of Pi results in conformational change (‘powerstroke’) and 8nm step size of dynein
How does 1 dynein gene regulate the transport of many, distinct cargo?
Dynein cannot function by itself, transport requires dynactin – a large complex linking dynein to cargo and regulating dynein activity
This complex can interact with a range of adaptor proteins, thus providing specificity for different cargo
How do bi-directional transport occur?
Different motors can associate with the same organelle or vesicle
Therefore, mechanisms exist to turn off one motor while turning on another, e.g. dynein vs. kinesin, to allow movement towards different regions of the cell
Especially important for long range movement in neurons
Our understanding of microtubule based movement comes from study of fish and frog melanophores
Melanophores are cells in the skin that contain melanin-filled pigment granules called melanosomes
Movement of pigment granules by microtubule motors
Melanosomes (pigment granules) are transported by kinesin-2 during dispersal, also tethered in the periphery by myosin actin motors (myosin V)
Dynein-dynactin motors are responsible for aggregation of melanosomes
Dispersion and aggregation are regulated by intracellular cAMP levels
cAMP activates downstream signalling via PKA, may influence motor protein activity/interactions
Why are protein targeting pathways needed?
Proteins are produced in the cytoplasm
Diffusion between membrane compartments is limited by lipid barriers
Protein transport is essential for organelle functioning
What is a protein targeting sequence?
It is a sequence of amino acids inside the proteins amino acid structure that tells the cell where to deliver the protein to
It tells a receptor protein where to deliver the protein to within the cell
What does the receptor protein do?
Receptor protein recognizes Protein targeting sequence
Transport complex binds Receptor-Cargo complex
Receptor-Cargo complex is imported into the compartment (targeted location, e.g. endoplasmic reticulum)
How are sorting signals used to control protein localisation?
In the example shown, the transfer of the ER signal sequence from the protein in the ER to the protein in the cytosol results in the two proteins locations being swapped out
How are sorting signals used to visualise organelles?
Endoplasmic reticulum - ER localization signal fused to red fluorescent protein mCherry
Peroxisome - peroxisome targeting sequence (PTS) fused to green fluorescent protein GFP
Protein targeting summary table
Nucleus overview
Nucleus is surrounded by a double membrane – nuclear envelope
Nuclear envelope contains Nuclear Pore Complexes for transport of molecules (proteins, mRNA, etc.)
Small molecules (<40kDa) can freely diffuse`
What are nuclear pore complexes?
NPC functions as a molecular sieve, limiting the free passage of macromolecules
NPC consists of 500-100 proteins of ~30 different Nucleoporins:
- (Nups, e.g. Nup53)
- Membrane, Scaffold, and Peripheral Nups
Central FG-Nups establish a barrier through unfolded Phenylalanine-Glycine repeats (~1/3 of all Nups)
FG-Nups interact with Nuclear Transport Receptors (Importins for import, Exportins for export)
What is a nuclear localisation signal? (NLS)
A nuclear localisation signal or sequence is an amino acid sequence that ‘tags’ a protein for import into the cell nucleus by nuclear transport
Classical NLS (cNLS) consists of a stretch of basic amino acids (Lysine (K) and Arginine (R))
cNLS can be mono- (SV40) or bipartite (Nucleoplasmin)
Another type is Proline-Tyrosine (PY)-NLS (e.g. nuclear heterogenous ribonucleoprotein hnRNP)
What are importins?
Nuclear transport is mediated by Importin (nuclear import receptor) and Exportin proteins
Importin alpha (α) recognizes and binds the nuclear localisation signal (NLS) and forms a trimeric complex with importin beta (β)
Importin β interacts with FG-Nups in the NPC to import cargo into the nucleus
What is Ran and what is the Ran GTP Cycle?
Conserved protein Ran is required for nuclear trafficking
Ran GTP-ase exists in two states – GTP-bound inside the nucleus and GDP bound outside the nucleus
Ran gradient drives nuclear import and export
How does Ran GTP cycle import proteins?
Importin α recognizes and binds NLS and forms a trimeric complex with importin β
Importin β interacts with FG-Nups in the NPC to import cargo into the nucleus
Nuclear Ran-GTP bind Importin releasing the cargo (Protein delivered into the nucleus!)
Ran-GTP complex with Importin is exported form the nucleus (with the help of exportin)
In the cytoplasm GTP is hydrolysed and receptor is released for next import
Protein targeting table summary for protein import from the cytosol into the nucleus?
Mitochondria protein synthesis location
Mitochondria contains 1500 proteins
99% of mitochondrial proteins are encoded by nuclear genes, synthesised on cytosolic ribosomes and imported into mitochondria
1% of mitochondrial proteins are encoded by the mitochondrial genome and synthesised in the mitochondrial matrix
What is the Mitochondrial Signal Sequence (MSS)?
99% of Mitochondrial Matrix Proteins are produced on cytoplasmic ribosomes and contain an N-terminal MSS (= MLS mitochondrial localization sequence)
N-terminal signal sequence is an amphiphilic alpha helix
Signal sequence is recognized by a membrane receptor
Proteins are imported post-translationally
Signal sequence is cleaved in the matrix
What are TOM complexes?
The TOM (translocase of the outer membrane) complex transfers proteins across the outer membrane
The TIM complexes (TIM23) transfer proteins across the inner membrane
What do proteins do to be able to enter the mitochondria?
Proteins unfold to enter mitochondria
- Import receptor of the TOM complex binds the signal sequence of mitochondrial precursor protein
- Unfolded protein passes through the translocation channel (TOM/TIM)
- Protein is translocated with the help of Hsp70 chaperones and the signal peptide is cleaved
- Signal peptidase cleaves the mitochondrial signal sequence (MSS) in the matrix
What drives protein translocation in mitochondria?
ATP hydrolysis and H+ drive protein translocation
Cytoplasmic (1) and mitochondrial (3) Hsp70 chaperones guide protein import inside mitochondria. Hsp70 Chaperones assist in protein folding and translocation hydrolyzing ATP in the process
Proton gradient helps drive protein translocation (2) through the TOM/TIM pore
Hsp60 chaperone helps fold the protein after translocation and N-terminal signal peptide cleavage
How are proteins translocated into the inner mitochondrial membrane? (pathway 1, not entering matrix)
Inner membrane protein has a hydrophobic sequence after N-terminal signal peptide that stops its translocation at the TIM23 complex
Signal sequence is cleaved and the protein remains inserted in the inner membrane
How are proteins translocated into the inner mitochondrial membrane? (pathway 1, entering matrix)
Inner membrane protein is delivered into the matrix through TOM/TIM complex, signal sequence is cleaved
A new N terminus is recognized by the OXA (oxidase assembly translocase) complex and protein is inserted into the inner membrane
How are proteins translocated into the mitochondrial inter membrane space?
One way is protein insertion into the inner membrane followed by cleavage of the transmembrane region
How are proteins translocated into the outer mitochondrial membrane?
Outer membrane protein (e.g. VDAC porin) is transported unfolded into intermembrane space by the TOM complex
SAM (sorting and assembly machinery) complex inserts the protein into OM and helps them fold
What are the mitochondrial protein translocation complexes?
Protein targeting summary table in the mitochondrial matrix
How does protein localisation to chloroplasts occur?
The TOC (translocase of the outer membrane of the chloroplast) complex transfers proteins across the outer membrane
The TIC complexes transfer proteins across the inner membrane
Signal peptide on the N-terminus with no well-defined sequence directs protein inside the chloroplasts
Import requires protein unfolding
ATP and GTP energy drives chloroplast import
TIC complex has internal motors that drive protein translocation
How does endoplasmic reticulum protein import occur?
ER protein import is a co-translational process
Membrane-bound ribosomes coat membranes of the rough ER
Protein is translated and imported into the ER at the same time – co-translational protein import
How was the ER signal sequence discovered?
Proteins were translated with and without ER present
Proteins translated without ER were slightly longer than proteins translated with ER present
This is because the ER sequence signal is retained without the ER
Discovered by Blobel and Sabatini, 1971
“proteins have intrinsic signals that govern their transport and localization in the cell”
What are the characteristics of the ER localisation sequence?
N-terminal signal sequence localizes proteins to the ER
ER-localised proteins have a core of ~8 hydrophobic amino acids preceded by one or more basic amino acids (R/K)
The mature protein in the ER loses the signal sequence by cleavage between the residues
Human preproinsulin -
MALWMRLLPLLALLALWGPDPAAAFVN
Bovine proalbumin -
MKWVTFISLLLFSSAYSRGV
Mouse antibody H chain -
MKVLSLLYLLTAIPHIMSDVQ
Chicken lysozyme -
MRSLLILVLCFLPKLAALGKVF
How is the ER signal sequence guided to the ER?
Signal Recognition Particle (SRP) is a protein-RNA complex
SRP binds to SRP receptors on the ER membrane
SRP binds ER signal sequence through a large hydrophobic pocket
SRP is a rodlike structure, which wraps around the large ribosomal subunit – temporarily blocks protein translation
Overview of SRP guidance to ER
SRP and SRP-receptor guide protein to the ER
SRP conformational change uses energy of GTP
What is Sec61?
The Sec61 complex is a transmembrane channel that spans the membrane of the endoplasmic reticulum
Sec61 translocon channel is gated by a short alpha helix (to keep the membrane impermeable to small molecules)
The channel opens transiently to translocate the polypeptide chain
Notice that the pore can also open sideways (important for transmembrane proteins and signal peptide release)
Does translocation into the ER lumen require energy?
Yes
SRP conformational change uses energy of GTP
How does translocation into the ER membrane occur?
N-terminal signal sequence is recognized by SRP and Sec61, transferring the chain into the lumen
An additional hydrophobic signal (Stop transfer sequence) stops translocation
Signal sequence is cleaved by signal peptidase
Lateral pore opening releases the protein into the membrane
Protein targeting summary table in the mitochondrial matrix
Where are lipid droplets formed?
LDs are produced from the outer layer of endorplasmic reticulum membrane
What are the two classes of lipid droplets?
Class 1
- LD proteins are imported through the ER
Class 2
- LD proteins are inserted post-translationally through amphipathic helices or lipid anchors
How do amphipathic helices target proteins to LDs
Amphipathic helices are alpha helices with specific distribution of charged or hydrophobic residues
Amphipatic helices preferentially partition into a less densely packed phospholipid monolayers
What are perxoisomes?
All eukaryotic cells contain peroxisomes
Peroxisomes synthesize essential lipids (e.g. ether lipids)
Peroxisomes catalyze reactions that pass electrons to oxygen producing peroxide
What is the peroxisome targeting signal 1? (PTS1)
The Peroxisomal Targeting Signal-1 (PTS1) is a C-terminal tripeptide that is sufficient to direct proteins into peroxisomes
[SAGCN]1-[RKH] 2-[LIVMAF] 3
PTS1 is localized at the very C-terminus
One PTS is sufficient to import the protein into peroxisomes
What is the peroxisome targeting signal 2? (PTS2)
Peroxisomal targeting signal-2 (PTS2) is an N-terminal amino acid sequence:
[RK]-[LVIQ]-X-X-[LVIHQ]-[LSGAK]-X-[QH]-[LAF]
PTS2 is localized close to the N-terminus
One PTS is sufficient to import the protein into peroxisomes
What is PEX5?
PEX5 is the receptor for PTS1 and PTS2
PEX5 interacts with the PTS signal via a series of tetratricopeptide repeats (TPRs*) within its C-terminal half
TRP are regions of repeated alpha helical 34aa motifs
What is PEX13 and PEX14?
PEX13 and PEX14 are subunits of a peroxisomal protein translocation complex
“PEX13/PEX14 Translocation Complex”
PEX5-Cargo complex binds PEX13/14 translocation complex to import cargo inside peroxisomes
PEX13 has YG repeats
What can peroxisomes import?
Peroxisomes can import large folded proteins, protein complexes, and even particles coated with PTS
Peroxisomal import does not require energy!
PEX5 recycling requires energy of ATP through PEX1/PEX6 ATPase
How does peroxisomal important occur? Model 1 NPC-like
Protein import into peroxisomes occurs through a nuclear pore-like phase
A cohesive YG rich domain of PEX13 forms a meshwork promoting PEX5-Cargo diffusion
Condensation peroxisomal import model
PEX5-cargo complex interacts with YG repeats of PEX13 inducing its clustering
YG repeats create a mesh like structure that helps import cargo bound to PEX5 inside peroxisomes
How does peroxisome export occur?
PEX5 receptor is exported through PEX2-10-12 complex
PEX5 binds Cargo that contains PTS
PEX5-Cargo complex interacts with YG repeats of PEX13 and passes into peroxisomes
Cargo dissociates; PEX5 is recycled in an unfolded state back to the cytoplasm
Peroxisomal import summary table in the mitochondrial matrix
What happens if PEX5 is lost in a developing brain?
Loss of PEX5 leads to an impaired peroxisomal import and a complete loss of peroxisomal functions – Peroxisome Biogenesis Disorder (PBD)
The Zellweger spectrum of peroxisome biogenesis disorders (PBD) are autosomal recessive disorders characterized by neurological defects, that include demyelination
What if a protein has both PTS and MLS?
Proteins can be localized to two or more different compartments through multiple targeting signals
Yeast phosphatase Ptc5 has an N-terminal mitochondrial localization sequence and C-terminal peroxisomal targeting sequence 1 (PTS1)
Can a protein without a targeting sequence be imported?
Proteins can enter peroxisomes through interaction with other peroxisomal cargoes – piggy backing
Malate dehydrogenase Mdh2 enters peroxisomes though Mdh3 interaction (Mdh3 has a PTS1)
What is planktonic and sessile bacteria?
‘planktonic’ – freely existing in bulk solution
‘sessile’ – attached to a surface or within a biofilm
No permanent connections with other cells but they can adhere to surfaces, food etc
What are the two main types of tissues?
Connective tissues (bones, tendons)
- Low cell density, abundant extracellular matrix
- Cell- cell contacts are rare
- ECM is load bearing
- Cell attachments to the ECM allowing force transmission
Epithelia tissues (gut lining, skin epidermis)
- Cells closely bound together into epithelia sheets
- Thin extracellular matrix on one face - basal lamina
What is the extracellular matrix?
The materials lying outside the cell are known collectively as the extracellular matrix (ECM)
Any material produced by cells and secreted into the surrounding medium
The ECM is a complex network of proteins and polysaccharide chains that are manufactured by cells, secreted and modified outside the cell by several different enzymes
What are the functions of the ECM?
Mechanical:
- Tensile and compressive strength and elasticity
Protection:
- Buffering against extracellular change and retention of water.
Organisation:
- Control of cell behaviour by binding of growth factors and interaction with cell-surface receptors.W
Where are extracellular matrix macromolecules secreted from?
In most connective tissues, the matrix macromolecules are secreted by cells called fibroblasts
What are the main macromolecular components of the ECM
Glycosaminoglycans (GAGs)
- Acidic polysaccharide derivatives, proteoglycans)
Fibrous proteins
- Includes members of the collagen family
Non-collagen glycoproteins - e.g. fibronectin and laminin
What are Glycosaminoglycans (GAGs)?
GAGs are unbranched polymers of repeated disaccharide derivatives, including amino sugars, sulfated acetylamino sugars and uronic acids.
Properties of GAGs?
Acidic and negatively charged
Attract positive ions (eg Na+) which attracts water causing gel formation
Comprise 10% of ECM mass but 90% of volume
GAGs (especially hyaluronan) provide compressive strength
Metabolically cheap bulking agent
What is Hyaluronan?
It is a type of GAG
N-acetyl-glucosamine- glucuronic acid
Hyaluronan is spun out from the cell membrane
Enormous (107 kDa - much larger than other GAGs)
Not sulfated
Not attached covalently to protein – ‘stand alone’
Often added to the ECM to hold open areas that would otherwise fill up with cells; it is then removed by hyaluronidase after appropriate cell migration
25,000 repeating units (sugar residues long)
Other GAG properties
Usually covalently bound into proteoglycans
20-200 sugars residues long
What is a proteoglycan?
A proteoglycan is a serine-rich protein decorated with hundreds of O-linked (usually via serine), acidic, sulfated GAGs
How are proteoglycans formed?
A specific link tetrasaccharide is first assembled on a serine side chain
The rest of the GAG chain, consisting mainly of a repeating disaccharide unit, is then synthesized, with one sugar being added at a time
What is aggrecan?
It is a proteoglycan
Its core protein is decorated with around 100 chondroitin and 30 keratan chains
Aggrecan then binds to hyaluronan (100:1) via adaptor proteins
Aggrecan-hyaluronan aggregates can be as big as bacteria (5 µm long)
What are Heparan sulphate proteoglycans
Heparan sulphate is a polymer of trisulphated GlcNAc and iduronic acid
Important role in cell growth
Bind chemokines at inflammatory sites, prolonging white-cell attracting activity
Bind and block certain proteases
Oligomerises FGF (fibroblast growth factor), giving easier binding to its tyrosine-kinase receptor
What is collagen?
Collagen - fibrous protein consisting of three α-chains forming a triple helix
Provides tensile strength to the ECM
Collagen chains consist of GXY repeats
G – Glycine
X – commonly proline
Y – commonly hydroxyproline
Hydrogen bonding between the -OH groups of hydroxyproline stabilises the triple helix
Lysines can be hydroxylated and subsequently glycosylated
Collagen synthesis (inside cell)
Occurs on RER
3x pro-alpha-chains come together to assemble a procollagen (via hydrogen bonding at y residues) each with a terminal propetide on each end (6 total)
Procollagen is secreted from vesicles into extracellular space
Collagen synthesis (outside cell)
Terminal propeptides are cleaved to form 100nm long collagen chains
Once its outside the cell, collagen molecules are crosslinked to form fibrils
Oxidative deamination of hydroxylysine and lysine forms
reactive aldehyde groups, which link molecules together
(and also link α-chains together too).
Collagen fibrils then self-assemble into fibres,
Collagen fibrils are highly stable and last around 10 years
(compare to most enzymes which turn-over in about an hour).
Types of collagen
Type I – Most common fibrillar form – found in skin bones and tendons
Type II – Similar tensile strength to cartilage
What is elastin?
Collagen gives the ECM its tensile strength, Elastin provides the ECM with elasticity
Elastin characteristics
- ~750 aa long
- Highly hydrophobic
- Rich in proline and glycine
- Non glycosylated
Alternating stretches of hydrophobic residues and alanine / lysine rich α helices
Crosslinking via α helical regions.
Hydrophobic domains are extensible due to loose random coil conformation
What does the basal lamina do?
The basal lamina plays several important roles
Structural
Determines cell polarity
Organises and binds cells
Forms a barrier to certain cells
Forms highways for cell migration
ECM in plants structure
The primary cell wall is flexible allowing cell expansion and remodelling
Multicomponent structure:
- Cellulose
- Hemicelluloses
- Pectins
- Arabinogalactans
- Cell wall proteins
The secondary cell wall is rigid providing strength
Multicomponent structure
- Cellulose
- Hemicellulose
- Lignin
Primary cell wall structure
Cellulose microfibril (made up of glucose linked at 1-4)
(1-4)Beta-D-glucan
Pectin
- Homogalacturonan
- Rhamnogalacturonan
- Arabinan
- Galactan
Hemicelluloses
Xyloglucan, xylan, glucomannan
Arabinoxylan, callose (1-3) -D-glucan
Structural
protein
Cellulose structure
Cellulose is composed of a polymer of glucose
Glucose comes together to form cellulose
Cellulose comes together to form microfibril
Microfibrils come together to form Fibril
Structure of hemicellulose?
Just glucose
Polysaccharide backbone with other sugars branching off of it
What is xyloglucans?
Most common type of hemicellulose
Xyloglucan can account for up to 20% of the primary wall dry weight.
Xyloglucan has a backbone composed of 1,4-linked β-D-Glcp residues.
Up to 75% of these residues are substituted at O6 with mono-, di-, or triglycosyl side chains.
Glucose backbone
What is pectin
Important for forming gel like structure of extracellular matrix
Can either be a monomer of galacturonic acid
Will always have a backbone of sugar containing acids
In some pectins you can have alternate sidechains
Pectin crosslinks to form macromolecular structures
Pectin methylesterases (PMEs) can demethylate pectin which is then available for crosslinking via Ca++ bridges
Secondary cell wall structure
Secondary cell walls predominantly cellulose, hemicellulose and lignin
S1, S2 and S3 layers have different orientations of the cellulose microfibrils
Secondary cell wall deposition results in a rigid cellular structure
The bacterial ECM strucutre?
The capsule is composed of high molecular weight polysaccharides
What is a bacterial ECM?
Some bacteria produce a biofilm
Matrix produced by the organism
Hydrated extracellular polymeric substances – polysaccharides, proteins, nucleic acids, lipids
Provide mechanical stability to the biofilm
Mediate cohesion to surfaces and form a network that immobilises the cells
Acts as an external digestive system – keeps extracellular enzymes in close proximity to cellsW
What are the 3 types of cell junctions?
- Occluding junctions: seal cells together into sheets (forming an impermeable barrier)
- Anchoring junctions: attach cells (and their cytoskeleton) to other cells or extracellular matrix (providing mechanical support)
- Communicating junctions: allow exchange of chemical/electrical information between cells
Junctions and epithelial function
More than 60% of cell types in vertebrates are epithelial
Epithelial function is to enclose and partition the animal body
Common organisation
– Anchored to the underlying tissue via the basal lamina on one side (basal)
- Free of attachment at the other side (apical)
Epithelial cells are polar
Act as selectively permeable barriers – fluid on the outside cannot readily permeate the epithelial membrane
Requires occluding junctions (vertebrates – tight junctions)
What are occluding junctions
Seals gaps between epithelia cells to create an impermeable or selectively permeable barrier
What are anchoring junctions?
Cell-cell adhesions and cell-matrix adhesions
Transmit stresses, tethered to cytoskeletal filaments inside the cell
What are the types of anchoring junctions?
Actin filament attachment sites:
- Cell –> Cell junctions = Adherens Junctions
- Cell –> Matrix junctions = Actin-linked cell matrix adhesions
Intermediate filament attachment sites:
- Cell –> Cell junctions = Desmosomes
- Cell –> Matrix junctions = Hemidesmosomes
What are adherens junctions?
Important role determining shape of multicellular structures
Form an indirect link between actin cytoskeletons
Allows coordination of cell activities`
Adhesion belts run longitudinally around the cell
Intercalated disks are regions of contractile cell attachment and function to co-ordinate the contraction of the heart
Adhesion belt reshaping of epithelial cells
The Adhesion Belt allows reshaping of sheets of epithelial cells
What are desmosomes junctions?
Similar to Adherens cell –> cell junctions but link to intermediate filaments, not actin
Provide mechanical strength
Found in vertebrates but absent in Drosophila
Present in mature vertebrate epithelia
What are channel forming junctions?
Create a link between cytoplasms of different cells
- Gap junctions (in animals)
- Plasmodesmata (in plants)
Gap junctions and plasmodesmata
Tight junctions block the passage of small molecules
Gap junctions in contrast create passageways between cells
Plasmodesmata in plants; gap junctions in animals
Allow exchange of small molecules between cells
Functions of gap junctions
In tissues with electrically excitable cells gap junctions couple cells allowing rapid spread of action potentials
Important in escape responses in fish and insects
Vertebrates – gap junctions in heart muscle and smooth muscle cells in the intestine synchronises contraction
Provide connections between cells allowing movement and coordinated sharing of signals
Eg liver cells allowing a response to signals from nerve terminals that contact only some cells
Structure of a Gap Junction
Complexes of protein subunits constitute
the gap junction channels
Either connexin or innexin subunits
Structurally similar but unrelated
Both present in vertebrates (mainly connexins)
Only innexins in Drosophila and Caenorhabditis
Each channel complex made of individual connexin subunits
Can be homomeric or heteromeric
Assemble into homotypic or heterotypic channels
What are signal relaying junctions
Allow signals to be relayed between cells across the plasma membranes at the site of cell-cell contact
Similar in principle to Channel-forming junctions
More complex structures
Typically include anchorage proteins alongside proteins
mediating signal transduction
Types:
- Synapses
- Immunological synpases
- Transmembrane cell-ligand cell-cell signalling contacts
