L14 - Vesicular transport 2 - adaptor proteins

what is the function of adaptor proteins?

link the membrane bound cargo to the coat

• they recognise and select cargo ensuring specificity

• different adaptor proteins recognise specific cargo motifs ensuring accurate trafficking

how do adaptor proteins recognise specific cargo motifs?

they do this by recognising signals in the cytoplasmic domains of transmembrane proteins

what is the family of 5 related adaptor complexes?

AP-1 to AP-5

• these are related as they have very similar structure and key roles in cargo selection

what makes up the adaptor protein (AP) subunits?

• made up of 4 subunits

• 2 large subunits (alpha, beta, gamma)

• 1 medium subunit (μ)

• 1 small subunit (sigma)

what do the large subunits have?

they have ear domains connected by flexible hinges

what is the subcellular localisation of the following adaptor complexes AP-1, AP-2 and AP-3 ?

• AP-1 = trans Golgi network

• AP-2 = plasma membrane

• AP-3 = trans Golgi network

why is subcellular localisation of adaptor complexes important?

they are targeted to the correct location to ensure the right cargo is selected and delivered to the right destination

what is the role of AP-2 at the pre-synaptic terminal?

• involved in synaptic vesicle recycling

• reforms synaptic vesicles after neurotransmitter release

what is the role of AP-2 at the post-synaptic terminal?

• involved in receptor endocytosis

• regulates synaptic plasticity

what do the medium and small subunits of AP-2 recognise?

these subunits recognise the sorting signals which are important for recognising the cargo

what is the importance of adaptors?

• to ensure the selectivity and accuracy of protein transport

• making sure the right cargo goes to the right destination

• cargo selection, sorting, trafficking

what is another family of proteins important for regulating vesicle transport?

Rab family of proteins - members of the Ras superfamily

what do the Rab family of proteins do?

• regulate specific trafficking steps

• they have distinct subcellular localisation - each Rab is associated with a particular organelle

• they cycle between the relevant membrane and the cytosol

• they are required for fusion in order for the right vesicle to fuse with the right target compartment

• act as molecular switches

where is Rab5 found and what is its function?

• at the cell surface, early endosome and clathrin

• role in early endosome fusion

where is Rab1 found?

Golgi complex

what are the steps of the Rab cycle?

1. Rab is bound to GDP and isolated in cytosol by GDI (GDP Dissociation Inhibitor) keeping it soluble

2. Rab is recruited to donor compartment through action of GEF which catalyses the exchange of GDP into GTP - activating Rab

3. Rab is now associated with a budding vesicle followed by budding off and formation of a transport vesicle

4. Transport vesicle then docks with the aid of a Rab effector

5. The SNARE complexes form along with membrane fusion

6. GAPs stimulate GTP hydrolysis

   RabGTP → RabGDP

7. Rab-GDP is then extracted from the membrane by GDI and recycled in the cytosol

what is a Rab effector?

• protein that specifically binds to the active GTP-bound form of Rab

what does the Rab effector do?

• helps the GTP-bound Rab carry out its functions

• it does this as it recognises Rab and helps the vesicle come closer to the target membrane to form the SNARE complex

what is one of the main types of effectors that bind to active Ras-GTP?

tethering proteins

what is the function of the tethering protein?

• the tethering proteins on the target membrane recognise the incoming vesicles and reach out to interact allowing it to dock

• the interaction between the tether and the activated Rab-GTP promotes:

  1. the closeness of the vesicle with the membrane

  2. helps fusion occur

what are the steps of the tethering protein (Rab effector)?

1. the tethering protein is recruited to the target membrane

2. the tether binds to active Rab-GTP on the incoming vesicles forming a bridge - bringing the vesicle close to the target membrane

3. vesicle and target membrane SNARE proteins interact - aligning the membranes tightly and preparing for fusion

4. the SNARE complex fully assembles - driving the membranes to merge and release vesicle cargo into the target membrane

what is Rab5?

the master regulator of early endocytic pathway

• functions in fusion, tethering, motility, uncoating and sorting

• its interactions with many different effectors allows it to do all of these functions

• it uses its switch function (GTP to GDP) to allow to switch these functions on and off

what does the Sec23 mutation cause?

• cranio lenticulo sutural dysplasia (tissue specific disease)

• mutation leads to abnormal ER to Golgi trafficking

• traffic jam within the ER

• as result large cargo proteins (like collagen) cannot enter the COPII coated vesicles

how do mutations in the Sec23 gene lead to skeletal craniofacial defects?

• Sec23 mutations impair ER to Golgi protein trafficking by disrupting COPII vesicle formation

• there’s a reduction in the packaging of components of the ECM that are needed for frontanel closure

• Sec23 is not effective at recruiting the Sec13/31 coat

• accumulation of proteins (like collagen) in the ER which leads to ER stress and contributes to the skeletal craniofacial defects of CLSD (disease)

as collagen is much larger what is it transported by?

• transported via specialised tubulo-vesicular carriers which have specialised packaging proteins

what’s choroideremia?

• causes blindness in men due to retinal degeneration

• defects in CHM gene on X chromosome

• mutations in REP1 lead to blindness

what’s the griscelli syndrome caused by?

mutations in Rab27a

albinism - delivery of melanin in melanocytes cannot happen

what do mutations in Rab7 cause?

Charcot-Marie-Tooth neuropathy type 2B