Lecture 13 - Cargo Transport Flashcards
Why is Cargo Transport in cells important?
Due to Highly complex environment of Eukaryotic cells, biochemical reactions are compartentalised to improve efficiency, hence components need to be transported between compartments.
Define the Four Major Cargo Transport Mechanisms in cells.
- Diffusion - small molecules (e.g., Ions) diffuse locally, whilst Large objects (500kDa) are confined and do not diffuse
- Advective Flows - cytoplasmic streaming currents, which are generated by cytoskeleton
- Filament Growth/Tip tracking - pathogens hijack host cytoskeleton to move within/between cells
- Motor Driven - transport of various cargo via motor proteins, which convert ATP Hydrolysis into mechanical force for movement along cytoskeleton tracks
Describe the distribution of cytosketal filaments within cells, and how it controls transport
- Microtubules - radial, polarised network emanating from perinuclear MTOC and reaching periphery
- Actin - dense meshwork of short, branched filaments at periphery, which are polarised towards membrane
Distribution - controls motor-dependent transport, with actin filaments and MTs coordinating to perform short/long range transport
What are the general properties of Motor Proteins?
(3 Points)
- Dimeric (bipedal)
- Processive - perform consecutive GTP Hydrolysis reactions (steps) without detachment
- Interact with cargo via C-terminal tails
What is the problem with the number of motor proteins? How is it Solved?
- Whilst many different motor proteins exist, the number of different types of cargo/compartment is significantly higher, therefore motor proteins must be able to bind multiple cargo types specifically
- Solved by the use of cargo adaptors
What are Cargo Adaptors?
Specific Proteins (Peripheral/Integral) or Lipids which allow motor-cargo interaction to occur, often via activation of motor from inactive an conformation
What are RabGTPases?
Small Monomeric GTPases that function as molecular switches to control movement of/between membrane-bound compartments
How do RabGTPases perform their function?
By recruiting effector proteins (i.e., motors) to compartments via direct/indirect interaction
What is the Function of GDP dissociation Inhibitor (GDI)?
- Prevents dissociation of GDP in absence of GEF, and shields lipid tails of RabGTPase from random membrane insertion
Describe the Structure of Myosin V?
(4 Points)
Consists of Dimer of 200kDa heavy chains, with each containing:
1. Head domain - contains motor domain (ATP/Actin-binding)
- Long “Neck” Lever arm - spans helical repeat of actin filament (36nm steps)
- Globular Tail doman - divided into two subdomains, binds to cargo
How does the Nucleotide bound to Myosin V motor domain impact its Acting-binding affinity?
(2 Points)
- ADP-bound/Nucleotide free - High Affinity binding
- ATP-bound - Low Affinity binding (i.e. release)
What is meant by the Head-tail interaction of Myosin V?
In absence of cargo/cargo adaptor, the GTDs fold back on motor domains, inactivating their ATPase activity (Inactive Conformation)
How do Cargo Adaptors activate Myosin V (Give Two Examples) ?
Cargo adaptors can activate Myosin V by disrupting the head-tail interaction either:
* Directly - e.g., Rab11 (receptor recycling)
* Indirectly - e.g., Rab27a via Melanophilin (Melanosome Transport)
How does Rab11-GTPase directly activate Myosin-V?
Rab11-GTP switch regions interact with Myosin V tail subdomain II, disrupting intramolecular interactions
How does Rab27a Indirectly Activate Myosin-V via Melanophilin?
(2 Points)
- Melanophilin contains multiple binding sites (e.g., Rab27, Myosin, Actin)
- Interacts with SDI, using an allosteric mechanism to alter conformation of SD2, destabilising head-tail electrostatic interactions
Yeast Budding requires highly polarised transport. What transport proteins are involved in this process?
(3 Points)
- Formin - polarises actin in direction of bud/daughter cell
- Myosin V (Myo2) - transports various cargo (mRNA, Organelles) into the budding cell using several different cargo-specific adaptors
- Cargo Adaptors - regulated by cellular location, and phase of cell cycle
(i) How is Myosin V (Myo2) linked to the cargo (i.e. Vacuole) before transport?
(ii) How is it released in the daughter cell
(1 Point // 2 Points)
(i) Vac17 serves as adaptor protein between Vac8 (Vacuole protein) and Myo2
(ii) Phosphorylation of Vac17 by Vps41 and Yck3 leads to degradation of Vac17 at PEST sites by calpain/proteasome
* This causes dissociation of Myo2, which can then transport cargo back into mother cell
Describe the Structure of Kinesin-1
- Heterotetramer, consisting of:
1. 2 heavy (KHC) - contains conserved 350aa motor domain
2. 2 light chains (KLC) - contain cargo-binding tetratricopeptide
(i) How is Kinesin-1 found when unbound to cargo?
(ii) How is it Activated?
(i) When Unbound Adopts autoinhibited fold, with KHC tail interacting with motor domain
(ii) Electrostatic Interactions between cargo adaptors and light chain disrupt head-tail interactions
What is the Structure of Dynein?
1.4 MDa complex (12 SU, 2x6 Polypeptides) consisting of:
* DHC - Heavy chain
* DIC - Intermediate chain
* DLIC - Light intermediate chain
* DLC - Light Chain
How do Dynein defects affect humans (give example)
Lead to Neurological disease (e.g., Perry Syndrome)
What is Dynactin? What is its Function?
- 1.1 MDa complex consisting of 23 SU of 11 different types
- Dynein requires Dynactin complex for its activation, allowing movement along MTs
Why are Activating Adaptors required for Dynein Activation?
Dynein-Dynactin Interaction is weak, hence activating receptors are required to stabilise the interaction and allow activation/cargo binding
What are the common features of Dynein Cargo Adaptors?
- Long coiled-coiled structures (often contain amphipathic helices, which mediate dimerisation)
- Bind to DLIC C-terminus, and to cargo-associated proteins
How do Activating Adaptors Stabilise the Dynein-Dynactin Interaction?
- Form coiled-coil that runs along length of Dynein-Dynactin interface, assistin dynactin to induce conformational change in Dynein
- This helps to align MTBDs so they can bind to MTs
Why does Dynein require activation to transport cargo?
In absence of cargo/dynactin, Dynein exists as “inactive phi particle” due to electrostatic interactions between AAA ATPase domains, as well as between binding regions of stalk
How do Bacteria Hijack the cytoskeleton to transport themselves?
Actin Microfilaments are recruited to the bacteria via the membrane protein ActA, which contains:
* Actin/ARP2/3 interaction domain
* Proline Rich repeat - recruits VASP, an activator of ARP2/3 complex (recruits cofilin and G-Actin)
