YEAST - the secretory pathway Flashcards
Why do eukaryotic cells need trafficking?
- So that proteins can be made more complex
- So that modifications can be made at particular sub-structures/environments
- Allows retrieval back to ‘resident’ compartments
- Allows sequential modifications
What is the endocytic pathway?
From the cell surface –> endosome/lysosome
What is the secretory pathway?
From the ER –> Golgi apparatus. The cargo then undergoes CONSTITUTIVE or REGULATED secretion
How are protein modifications made – what is added where and then where are proteins taken for further processing?
Oligosaccharides are added to proteins in the ER and then the protein is further modified in different cisternae of the Golgi.
What parts of proteins does glycosylation occur on — N and O linked
N – at the asparganine amino acid.
O – at the O group found on SERINE and THREONINE groups
What is the purpose of glycoslyation?
- Allows correct folding
- Adds complexity to proteins
- Allows proteins to behave as ligands
- Aids protein interactions with the ECM
Examples of glycosylation
- Blood types
- Form of muscular dystrophy where glycosylation doesn’t occur on DYSTROGLYCAN protein
Process of glycosylation - where does it occur ?
Proteins are pulled into the ER where PRE MADE SUGARS are added.
Then different modifications (eg diff sugars added and proteins trimmed) are made at different cisternae of the Golgi. These add important properties to the protein
Why are simple models better in investigation of secretion?
Secretion occurs at a cellular level - therefore YEAST is a good model
Advs and Disadvs of yeast as a model
+
Simple, cheap, single-celled, grown in v large quantities, genome fully sequenced, pathways are conserved
- V tiny (ltd with microscopy), has a CELL WALL, limited genetic diversity
Why are some types of muscular dystrophy caused by defects in glycosylation?
DYSTROGLYCAN - sugar which links dystrophin protein to the ECM around MUSCLE CELLS
Sugars aid in this interaction
KEY YEAST STUDY
Main steps
1) Yeast mutagenised by temperature increase (to disrupt genes involved in the secretory pathway)
2) Most and least dense cells separated by fractionation – idea is that cells that are more dense have their secretory pathway mutated as there is a higher % of vesicles in the cell
3) Cells with 5% increase in density separated from WT cells and taken for further testing
HOW ARE DENSE MUTANT CELLS ANALYSED
- electron microscopy?
- acid phosphatase?
Use of electron microscopy to analyse the GLOBAL STRUCTURE of cells
Also measure cells ability to secrete products such as acid phosphatase
What was the overall finding of the yeast study?
Mutated cells —- different ULTRA STRUCTURE
Path of Protein stages in the secretory pathway?
1) pre pro form (before entering pathway)
2) gains sugars (ER)
3) protein modifications made (Golgi)
4) Secreted as 4 SHORTER PEPTIDES
How is western blotting useful in the context of investigating proteins in the secretory pathway?
Western blotting allows us to see which stage of the secretory pathway the protein is in
Analysis of results of yeast assay - what genes were identified and how were they classified?
23 genes identified as being needed for the steps in the secretory pathway
Group classified into A, B, C, D, E based on what proteins were mutated in the stages of the secretory pathway
Why weren’t ALL secretory proteins identified in the yeast assay?
Not all mutations induced by use of temperature (only temp sensitive mutants were impacted)
The assay only considered secretion to the PLASMA MEMBRANE, not the ENDOSOME
Where can a protein be trafficked to if not to the plasma membrane for secretion?
To the late endosome/ lysosome
