The secretory pathway Flashcards
Why do Eukaryotic cells need membrane trafficking?
- Compartmentalisation=complexity
- Specific subset of proteins depending on environment
- Modifications
- Retrieval
2 types of membrane trafficking pathways and where do they occur
- Secretory (biosynthetic)- making things constituency (happen without markers)
- er to Golgi to pm/endosome/lysosomes - Endocytic
- cell surface to Endosome to ER/Golgi
Stages of Membrane trafficking
- Protein synthesis of ribosomes transport of proteins across/into ER
- Budding and fusion of ER to Golgi
- Retrograde Golgi to ER
- cisternal progress (cics Golgi)
- Retrograde trafficking later to earlier Golgi cisterns
- Constituative secretion
- Regulated secretion
- Sorting to lysosomes
- Endocytosis
What studies showed biochemical compartmentalisation
By modification of various molecules - insulin - VSV-glycoprotein Used as already know Insect cells with virus to look at
How are proteins modified
Oligosaccharides added in ER and processed by Golgi
Gylcosylation- where it happens/how it works
N and O linked at proteins
Protein in ER- sugar added on and trim down
Golgi- more trimming and things added on
Purpose of glycosylstion
Change shape-To assist folding (ligand)
Intracellular- trafficking/sorting
Extracellular- interaction with exon
Importance of glycosylation in disease
- 0 linked sugars are the basis of ABO system of blood
- Muscular dystrophy- absence of sugars on the protein dystrolycon
- Staphylococcus aureus toxin binds static acid reside to glycosylated protein
What is dystroglcyon?
Links dystrophin to the extracellular matrix around muscle cells
Sugars facilitate interaction
Identifying the genes and proteins involved in the secretory pathway with a genetic approach
Organisms- mouse, zebrafish, c elegans, drosophila
Modal suitability- simplicity, specificity of secretion
Different organisms reveal different info
Yeast model advantages
Amenable
Genetics known
Cheap and easy
Pathways conserved
Yeast model disadvantages
Limited cell to cell
Limited gene diversity
Small
Cell wall
The key experiment using yeast to identify genes involved with the secretory pathway
- Navick and schekman 1989
- if vesicles couldn’t be secreted- cells were secretory deficient (sec) and cells would increase in density as these vesicles accumulate
- cells mutagenised and shifted to restrictive growth temperature (37)
- cells were fractioned in a gradient forming medium
Results of navick and schekman
5% increase in density of sec mutants (deficiency in secreting vesicles)
- complete separation of WT cells
How was the yeast experiment analysed and what were they looking for
Electron microscopy
- accumulation of vesicles could be observed
-proteins detected with different modifications through secretory pathway
Looking for global defects in secretion
Definition of secretory mutants and how it was defined
Strains which fail to export active invertase and acid phosphates but continue to synthesise proteins
- found due to ability to secrete invertase and acids phosphatase at permissive and restrictive temperature
How are Delta factor and CPY modified through the secretory pathway
Delta factor
Delta=prrpreform, core form gets sugars and is modified through the ER and golgi
- Late golgi chops to Ke2= 4 peptides secreted
- secreted MaF
CPY mutant form
- shows different size on western blot which shows where the protein is stuck
- vacuole Mcpy
What is redundancy
When other things compensate
What genes were identified from this experiment and what does this say about secretion?
23 identified
Required for transport from the ER to plasma membrane
Sec genes- 5 classes by combining mutants from different classes and by use of more detailed analysis of protein modification
What did novick and schekman find?
Secretory proteins move cytosol to ER to Golgi by vesicles to plasma membrane
Why weren’t all the genes/proteins involved in the exocytic pathway identified by novick and schekman?
- only identified temperature sensitive mutants
- only considered secretion to PM not endoscope or vacuole defects identified
- any redundancy functioning genes wouldn’t be identified
Vacuolar/lysosomal protein sorting function?
degradation of extracellular material taken up by endocytosis
Intracellular components by autophagy
Why is vacuolar/ lysosomal protein sorting important?
Organelles contain degradative/proteolytic enzymes that must be kept separate from the rest of the cell
Where are lysosome resident and ate golgi enzymes transported
Lysosome- thought secretory pathway to lysosome
Late golgi- sorted into pathway to lysosomes NOT pm
VPS
Vacuolar protein sorting screens
carboxyl peptide y= transported lysosome from golgi
What happened when labs mutagenised cells which secreted CPY
Did this using colour assay a vacuolar enzyme
- mutagen secreted enzyme rather than keeping it
The cells which secrete CPY are further analysed, How and what did they find?
microscope based and biochemical techniques
- genes identified and fall into distinct groups
How many CPYs were identified
60
combined to determine order of action
Types of analysis
- fluorescence microscopy- look at vacuole= white
- Light- some mutant material all over surface
- electron microscopy- mutant smaller
CPY western blot
propeptide stops activating until its in the right position
golgi add more man
vacuole reached, cleavage more mature form
How are vacuolar mutants divided into classes
Dependent on the stage at which they appear to block the route of the vacuole
Can be blocked trans, late, early and MVB
4 destinations things can be trafficked from the late golgi
- to plasma membrane
- early endoscope
- late endosome or MVB
- vacuole
What is trafficking from the golgi to late endosome?
CPY pathway
- synthesised in prepro from and is transported from ER to golgi
- sorting at golgi
How is CPY pathway sorted at the golgi and transported at late endosome
CPY specifically recognised by VPS10 receptor
CPY dissociates from VPS10 at the at late endosome and transported to the vacuole to mature (cleaved)
What does the transport step in the CPY pathway require
Cytoplasmic factors
- Clathrin
- 2 adaptors
What is the pathway from the GOLGI TO VACUOLE
Alkaline phosphatase pathway
What is trafficked in the ALP pathway
ALP and Vam3 traffick bypassing endosomes
- Alp= proteins in cytosol form recognition motif
- Vam3- claps up delta, use cathrin
Pathway to the early endosome
Kex2 pathway
making pheromone processing enzyme
involves clathrin
What does Kex2 pathway do?
signals retreival from late endsosome
can be phosphorylated
What is endocytosis?
Pm invaginates into the cells causing a vesicle to be made that is able to fuse with endosomes and enter endolysosomal membrane systems
Why do we need endocytosis?
- Retreival
- downregulation of signal
- remodelling of cell surface lipids and protein composition
- to allow things to enter the cell- pathogens and toxins
How do we know that there are multiple compatments involved in trafficking and it doesnt just go straight to the lysosomes?
- light follows movement= live cells
- electron reveals morphological differences in compartments
- Genetic studies- mutants block specific stages and no longer works
- separated on density- Early/late endosome
What are end-screens?
visualising endocytosis in yeast
- add dye
- taken into vacuole
- mutant cant take up
- internalise a fluid marker (lucifer yellow)
What does the time series of endo screens show?
Sequential localisation of actin to clathrin in patches of yeast
- clathrin and actin binding protein= CLC- GFP
- later stage move inward accompanied by actin (pushing force)- form filaments to push and grow
2 key features of the endocytic pathway model
- sequential assembly and dissembly of complexes involved in endocytosis
- the actin cytoskeleton plays a key role in inward movement of vesicles
Where are cargo proteins thought to be recruited?
At early stages of the process
by proteins that interest with that vesicle coat
The endosomal compartment
After internalisation in an endocytic vesicle it shows the protein pass through at least 2 distinct internal compartments with different densities
Late and early endosomes
-After this trafficked to vacuole for degradation
What is the late endosome? (MVB)
Key compartment with material entering from the exo and endo pathways
more mutants defective in vacuolar protein sorting are also defective in late stages of the endocytic pathway
Sorting in late endosome /MVB- pathway intersect
- Ste2- pherone receptor localises to PM, ubiquinated and internalised
- carboxypeptidase (CPS)- trafficked from golgi to LE/MVB to vacuole
- Vps10 (CPY receptor)- trafficked to LE /mvb- retreived
What do all 3 of these interecting pathway proteins do?
accumulate at the late endosome in class E mutants suggesting they are all defective in forming and sorting proteins to the lumental MVB vesicles - VPS class E mutant proteins associate into 1/4 complexes- ESCRT (Endosomal sorting complexes retrieved for transport)
