Lecture 13 - Cargo Transport

Question 1

Why is Cargo Transport in cells important?

Answer 1

Due to Highly complex environment of Eukaryotic cells, biochemical reactions are compartentalised to improve efficiency, hence components need to be transported between compartments.

Question 2

Define the Four Major Cargo Transport Mechanisms in cells.

Answer 2

1. Diffusion - small molecules (e.g., Ions) diffuse locally, whilst Large objects (500kDa) are confined and do not diffuse
2. Advective Flows - cytoplasmic streaming currents, which are generated by cytoskeleton
3. Filament Growth/Tip tracking - pathogens hijack host cytoskeleton to move within/between cells
4. Motor Driven - transport of various cargo via motor proteins, which convert ATP Hydrolysis into mechanical force for movement along cytoskeleton tracks

Question 3

Describe the distribution of cytosketal filaments within cells, and how it controls transport

Answer 3

• 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

Question 4

What are the general properties of Motor Proteins?

(3 Points)

Answer 4

• Dimeric (bipedal)
• Processive - perform consecutive GTP Hydrolysis reactions (steps) without detachment
• Interact with cargo via C-terminal tails

Question 5

What is the problem with the number of motor proteins? How is it Solved?

Answer 5

• 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

Question 6

What are Cargo Adaptors?

Answer 6

Specific Proteins (Peripheral/Integral) or Lipids which allow motor-cargo interaction to occur, often via activation of motor from inactive an conformation

Question 7

What are RabGTPases?

Answer 7

Small Monomeric GTPases that function as molecular switches to control movement of/between membrane-bound compartments

Question 8

How do RabGTPases perform their function?

Answer 8

By recruiting effector proteins (i.e., motors) to compartments via direct/indirect interaction

Question 9

What is the Function of GDP dissociation Inhibitor (GDI)?

Answer 9

• Prevents dissociation of GDP in absence of GEF, and shields lipid tails of RabGTPase from random membrane insertion

Question 10

Describe the Structure of Myosin V?

(4 Points)

Answer 10

Consists of Dimer of 200kDa heavy chains, with each containing:
1. Head domain - contains motor domain (ATP/Actin-binding)

2. Long “Neck” Lever arm - spans helical repeat of actin filament (36nm steps)
3. Globular Tail doman - divided into two subdomains, binds to cargo

Question 11

How does the Nucleotide bound to Myosin V motor domain impact its Acting-binding affinity?

(2 Points)

Answer 11

• ADP-bound/Nucleotide free - High Affinity binding
• ATP-bound - Low Affinity binding (i.e. release)

Question 12

What is meant by the Head-tail interaction of Myosin V?

Answer 12

In absence of cargo/cargo adaptor, the GTDs fold back on motor domains, inactivating their ATPase activity (Inactive Conformation)

Question 13

How do Cargo Adaptors activate Myosin V (Give Two Examples) ?

Answer 13

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)

Question 14

How does Rab11-GTPase directly activate Myosin-V?

Answer 14

Rab11-GTP switch regions interact with Myosin V tail subdomain II, disrupting intramolecular interactions

Question 15

How does Rab27a Indirectly Activate Myosin-V via Melanophilin?

(2 Points)

Answer 15

• 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

Question 16

Yeast Budding requires highly polarised transport. What transport proteins are involved in this process?

(3 Points)

Answer 16

• 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

Question 17

(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)

Answer 17

(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

Question 18

Describe the Structure of Kinesin-1

Answer 18

• Heterotetramer, consisting of:
  1. 2 heavy (KHC) - contains conserved 350aa motor domain
  2. 2 light chains (KLC) - contain cargo-binding tetratricopeptide

Question 19

(i) How is Kinesin-1 found when unbound to cargo?
(ii) How is it Activated?

Answer 19

(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

Question 20

What is the Structure of Dynein?

Answer 20

1.4 MDa complex (12 SU, 2x6 Polypeptides) consisting of:
* DHC - Heavy chain
* DIC - Intermediate chain
* DLIC - Light intermediate chain
* DLC - Light Chain

Question 21

How do Dynein defects affect humans (give example)

Answer 21

Lead to Neurological disease (e.g., Perry Syndrome)

Question 22

What is Dynactin? What is its Function?

Answer 22

• 1.1 MDa complex consisting of 23 SU of 11 different types
• Dynein requires Dynactin complex for its activation, allowing movement along MTs

Question 23

Why are Activating Adaptors required for Dynein Activation?

Answer 23

Dynein-Dynactin Interaction is weak, hence activating receptors are required to stabilise the interaction and allow activation/cargo binding

Question 24

What are the common features of Dynein Cargo Adaptors?

Answer 24

• Long coiled-coiled structures (often contain amphipathic helices, which mediate dimerisation)
• Bind to DLIC C-terminus, and to cargo-associated proteins

Question 25

How do Activating Adaptors Stabilise the Dynein-Dynactin Interaction?

Answer 25

• 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

Question 26

Why does Dynein require activation to transport cargo?

Answer 26

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

Question 27

How do Bacteria Hijack the cytoskeleton to transport themselves?

Answer 27

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)