Transport by vesicles1. Overview about vesicular transport in the cell2. Basics of vesicular transport3. How is target specificity achieved?

Compartments that communicate by vesicular transport

Vesicular transport

Vesicular traffic in the cell

1. Secretory pathway: major outward traffic (ER Golgi cell surface) 2. Endocytotic pathway: major inward traffic: (cell surface endosome lysosome)3. Retrieval pathways (Endosomes Golgi, endosomes cell surface, Golgi ER)

Main phases of vesicular transport

1. Budding2. Transport3. Docking4. Fusion

budding

transport docking fusion

donorcompartment

targetcompartment

The orientation of the membrane is preserved during budding and fusion events.

Lumenina of cell compartents that communicate with vesiclesare equivalent to the cell exterior

What do vesicles transport- beside cargo?

budding

transport docking fusion

donorcompartment

targetcompartment

cargo

budding

transport docking fusion

donorcompartment

targetcompartment

cargo

Vesicles transport

1. Cargo2.3.

budding

transport docking fusion

donorcompartment

targetcompartment

cargo

Vesicles transport

1. Cargo2. Membrane (lipids and proteins)3. Fluid (water, ions small molecules)

membrane fluid

1. Vesicle budding

Y

Y

Y YCargo receptors concentrate specific molecules

• selection of cargo

cargo

1. Vesicle budding

• Selection of cargo • Formation of vesicle

•Clathrin is connected to the membrane by adaptor proteins that bind to cargo receptors •Adaptin is a adaptor protein-complex (AP) made of four different proteins (adaptins).•Humans have four different AP complexes•Adaptins can influence the cargo specificity of a vesicle through their interaction with cargo receptors

Vesicle budding is driven by the assembly of a protein coat

Coated pit

Endocytotic vesicle form with the coat protein clathrin.

Clathrin is a three-legged protein (triskelion) that assembles into a cage-like structure

Dynamin forms a collar-like structure that constricts to ‘pinch off’ vesicles. (requires hydrolysis of GTP)

Defectiv Dynamin blocks vesicle formation at the plasma membrane.

Different types of coated vesicles

2. Vesicle transport

• Movement along actin fibers or microtubuli• requires interaction with motor proteins

3. Vesicle docking

TetheringVesicles are loosely bound to the target membrane (> 25 nm)Mediated by tethers proteins

DockingVesicles are more tightly bound to target membrane (<1.5 nm)Mediated by SNARE proteins

Whyte, J. R. C. et al. J Cell Sci 2002;115:2627-2637

tethering docking

Docking of vesicles is mediated by tethering factors (Tethers)

a) Long coiled-coil proteinsb) Large multi-subunit complexes

• Tethers can span relatively long distances (>200 nm) between thevesicle and the acceptor membrane.

• They may provide a molecular net to catch ‘relevant vesicles’• Tethers have a specific subcellular localization• Tethers interact with SNAREs and with Rabs

Whyte, J. R. C. et al. J Cell Sci 2002;115:2627-2637

“SNAREs: engines for membrane fusion”

1. Docking: close contact of the two membranes (< 1.5 nm)2. Membrane fusion

• requires displacement of water from the hydrophilic surfaces of the lipid layers (energetic unfavourable)

• Reaction is driven by the spontaneously assembly of SNAREs into a four helical bundle

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) |

A complex of four different SNAREs drives membrane fusion

After fusion SNARE complexes are disassembled for re-use.

Phases of vesicular transport

1. BuddingSelection of cargo and accessory proteins

2. Transport Movement along actin fibers or microtubulirequires interaction with motor proteins

3. Tethering Vesicles are loosely bound to the target membrane by tether factors

4. DockingVesicles are more tightly bound to target membrane by SNARE proteins

5. Fusionmight depend on a signal, (e.g. synaptic vesicles in neuron, insulin secretion)requires specialized protein machinery (SNARE) to overcome energetic barrier

Transport by vesicles1. Overview about vesicular transport in the cell2. Basics of vesicular transport3. How is target specificity achieved?

SNAREs contribute to target specificity.

Jahn and Scheller Nature Reviews Molecular Cell Biology 7, 631–643 (2006) | doi:10.1038/nrm2002

The assignment of SNAREs to membrane-trafficking pathways

The Rab protein family plays an important role in regulating all phases of vesicular transport

Rab proteins are molecular switches (active / inactive).

Active Rab can bind to other proteins and regulate their specific functions ( Rab effectors)

1. Cargo receptors potential role in protein sorting

2. Motor and motor adaptor proteins transport of vesicles

3. Tether factors tethering

4. SNAREs docking, fusion

• There are >60 different Rabs in mammalian cells.• Rabs are localized to specific compartments in the cell.

?

Rab proteins are small GTPases that function as molecular switches

Rab-GDP GDP GTP Rab-GTPGAP

GEF

GAP: GTPase activating proteinGEF: Guanine nucleotide exchange factor

“activated state”Interaction with effectors

“inactive state”

Effectors are proteins that selectively bind to the activated Rab (Rab-GTP) and mediates downstream effects.

Summary

• Protein transport between the ER and the Golgi apparatus and from the Golgi apparatus to other destinations (endosomes, plasma membrane) is mediated by vesicular transport

• Vesiclular transport occurs in different phases: budding, transport, tethering, docking and fusion

• Budding transport vesicles have distinct coat proteins on theircytoplasmic surface. The coat is linked to cargo receptors by adaptor proteins.

• SNARE proteins spontaneously assemble into four helical complexes that drive fusion of vesicle and target membrane

• Rabs are molecular switches that via their effectors regulate all phases of vesicular transport

• Rab effectors are: Cargo receptors, motor proteins and their adaptors, tethering factors and SNAREs