L1: Protein Sorting in the Endomembrane system: The secretory pathway

Why do we need protein exchange and sorting

• variety of organelles

  • each with specific

    • ion transport, signal transduction

    • biosynthetic processes

• need to maintain their protein compositions

  • even though they are constantly exchaning membrane lipids and proteins

• So need this sorting to generate and to maintain compartment identity

Em thin section through a monkey pancreas exocrine cell

What are the defined routes

1. Secretory (talked about in this lecture) (aka biosynthetic pathway)

   • takes newly synthesied protein

     • ER→ Golgi→ Plasma membrane

2. Endocytotic

   • first: internalisation of extracellular material (endocytosis)

   • then: series of endosomal compartment to the lysosome (or vacuole in fungi and plants)

Are they completely separate pathways?

No

• connected via tran-sgolgi network and endosomes

• many tracfficking routes are bi-diretional

  • anterograde and retrograde transport

Why are many trafficking route bi-directional

1. Maintain membrane homeostasis

• must balance retro and anterograde traffic

• other wise ER would get smaller and smaller

2. Help with the recycling of the protein

How can you map the secretory pathway: 1

• Pulse-Chase Approach

• Palade

How does the pulse-chase approach work and what did it show

1. pancreatic cells incubated with radioactive amino acids for a few mins→ pulse

2. Cells then incubated in unlabelled medium for variable lengths of time

3. Analyse by

   • autoradiography

   • EM

Results:

1. Labelled proteins detectable in the ER

2. then the Golgi

3. then the secretory granules

Therefore: shows the pathway/ mapping the secretory pathway

The graph shows the autoradiography graph

• shows how as decreases from Er→ goes to Golgi and then eventually to the seretory granules

EM of H³-Leucine

New way of mapping the secretory pathway by pulse chase

Live cell imaging of trafficking→ visualsing the movement as more of a wave than just at fixed time points

• Use fluorescent proteins→ GFP

or

• Use fluorescent lipids

How to create a GFP pulse to map the secretory pathway

1. Modified GFP multimerised with multimerisation factor in ER→ stops it from leaving the ER→no export

2. Next, borken up by Drug-induced monomerisation or temp shift to allow folding or transport releases GFP ‘wave’

3. allows the proteins to move further in the cell→ can then be visualised as a wave

Strucuture of the endoplasmic reticulum

Two functionally and morpholgically distinct domains

1. Rough

   • sheet-like

   • studded with protein-synthesisng ribosomes

2. Smooth

   • tubular

Functions of endoplasmic reticulum

1. Main site of protein biosynthesis

2. Protein folding

   1. general

   2. with S-S bonds

3. Post-translational modifications→ Protein Glycolysation

4. Quality control

2. Protein folding

General folding

• need chaperone to fold→ Binding Protein ‘BiP’ (e.g Hsp70)

• folds and prevents aggregation

Proteins with S-S

• In the cytosol→ reducing environemnt→ the S-S forms and folds the protein find

• In the cell→ oxidising comparment

  • sulfydryl groups on cystine reduces

  • and forms covaneltn bonds

  • assisted by→ Protein disulfide isomerases (PDI)

    • helps to cut and rejoin the S-S once it is in a new environement

3. Glycosylated

1. invariant glycan is transferred to asparagines (N) of the consensus NXS/T

   • note: X is any amino acid except proline S and T threonine

2. transferred in a single step

3. What does glycosylation help do

1. folding

   • glycan is hydrophilic and so ensures that the hydrophobic residues are folded into the inside of the protein

2. further assists chaperone binding

   • e.g calnexin

3. Signals the next step→ quality control

4. Quality control

• ensures only correctly folded proteins are shipped to the golgi

• what happens to the others:

  • exported to the cytosol→ degraded

ER export

1. Protein made in the rough ER

2. Moves to the smooth ER

3. Move to the specialised ER exit sites

4. Cargo is selectively packed into COPII (coat protein II) vesicles

5. COPII homotypic fusion (fuse with each other)

6. form vesicular tubular clusters (VTCs)

7. These can subsequently undergo further homotypic fusion events

8. RETROGRADE: This is the first compartment for recyling proteins

   1. proteins that escaped from the ER

   2. trafficking machinery itself

9. This is with COPI vesicles

What are the vesicular tubular clusters (VTCs) also known as

ER-Golgi intermediate compartment (ERGIC)

What happens next for the ERGIC itself

1. becomes attached to MT via dynein motors

2. Pulled towards the golgi

3. ERGIC may either

   1. fuse with an existing cis-cisterna

   2. undergo homotypic fusion to form new cis-cisterna

note: this depends on the two models see after

4. SECOND RETROGRADE: COP1 from cis-golgi back to the ER

Golgi Apparatus what is it

• Found next to the nuclear envelope

• stack of flattened fenestrated (with holes) membrane sacks (cisternae)

  • each with its own lumen

  • number of cisternae→ variable

    • depends on how much protein secretion the cell does

    • 6 or 3 or 20

• Have polarity:

  • Cis→ medial→ trans

    • cis (closest to the nuclear envelope)

  • with different functions and partly distinct protein and lipid complements

In which direction do the proteins traverse the golgi

• cis to tans direction

What is the cis-golgi-network

• area near the cis-most cisterna

• where ERGICs fuse

  • Cis and trans are network

  • cis is more tubular

  • with fenestrations

    • transport and fasiculations

  • no network in the medial cisternae

What is the point of going through the golgi

1. Post-translational protein modification

   • maturation and sorting

What modifications take place

1. trimming

2. addition or extension of attached glycans

3. sugar phosphorylation

4. proteolytic cleavage

In order to do this→ golgi have three functional domains

1. Cis

   • remove Manose

2. Medial

   • - Man, + GlcNAc

3. Trans

   • +Gal, + Sialic acid, Sulphation

enzymes found in these compartments are different and used to monitor the progress of secretory proteins

Other functions of golgi

Synthesis of

1. extracellular polysaccharides

2. glycosaminoglycans

3. plant and fungal cell wall polysaccharides

Two models for intra Golgi Transport

1. Vesicular transport model

   • vesicles bud from each compartment to the next

   • cargo: cis→ trans

2. Cisternal Maturation Model

   • Proteins arrive in tubular cluster

   • fuse together

   • make the newest cisternae

   • Golgi cisternae mature

     • exchange their protein complements over time

       • whilst the cargo always stays in the same cisterna

   • trans cisternae becomes vesicles and proteins taken further on

     • cargo: trans→ cis

both models assume vesciular tranport between cisternae

1. Vesciular tranpsort model EVIDENCE

1. EM showing vesciles budding from the edges of Golgi cisternae

2. in vitro transport assays

2. Cisternae Maturation Model EVIDENCE

1. Explains how cargo that is too big, e.g procollagen for vesciles can still be transported through

2. Live cell imaging in yeast

2. EVIDENCE: PULSE CHASE live cell imaging in yeast→ Why use yeast

• Mammalian cells→ golgi compartments are too close together

• Yeast→ three compartments are separated far enough as they do not stack

2. EVIDENCE for cisternal maturation model: PULSE CHASE

Procedure:

1. Label cis/medial golgi: GFP

2. Trans Golgi: RFP

Expected observations:

• If vesicular transport→ Static colours

• If Maturation→ conversion Green to Red

Result:

• Conversion green to red

• therefore Maturation model is correct

So which model is correct

• evidence for the maturation

• HOWEVER→ still may be some antereograde traffic still occur

  • no consesus but probable that both models are partly correct

What is the Trans Golgi Network (TGN)

• the cluster of tubules and vesicles at the trans-most side of the golgi

Is it a stable comparment?

• Yes→ it has characteristics of a stable comparment

• No→ In the cisternal maturation model→ corresponds to a trans-cisterna that is being converted into secretory carriers

What other function does it have in some species?

• endosome

Role of the TGN

Major protein sorting station→ Point at which proteins diverge for the first time

How are proetins sorted by the TGN

1. Bulk or default→ directly to plasma membrane.

   • may require no further sorting signals

2. Sorting into different carriers (direct secretion in polarised cells)

3. Concentrated in regulated secretory vesicles/ secretory granules

4. Endosome transport

1. Bulk transport→ live imaging shows how this secretion is mediated

• Tubular carriers are pulled out of the TGN by molecular motors on microtubules

• do not know about protein coat or other details

2. Sorting into different carriers (direct secretion in polarised cells)

• Apical vs basal

• Axon vs dendrites

  • have very different protein and lipid compositions

• Sort proteins into destined vesicles for certain places

2. Example of sorting

e.g Vesicular stomatitis virus envelope glycoprotein (VSV-G)

• into basal Sorting into different carriers (direct secretion in polarised cells laterally-destined vesicles

2. Investigating the secretion of VSVG with GFP

• shows that it is temperature dependent

• At a low temperature→ there is a TGN block

3. Regulated secretion and examples

• special carriers only fuse with the plasma membrane in response to the appropriate signal

  • e.g→ insulin release in pancreatic cells

  • e.g→ NT release in nerve cells

4. Endosome transport

• Clathrin-coated or other types of non-clathrin coated

• delivered to endosomes

  • works an an interface between the secretory and endocytotic pathways