Lecture 13 Yeast screens Flashcards
Why do eukaryotic cells need membrane trafficking?
Organelles are membrane bound
Compartmentalisation allows more complexity.
Enzymes can modify specific subsets of proteins in certain environments e.g glycosylation and proteolytic cleavage.
For sequential modifications, proteins need to be exposed to distinct sets of enzymes.
Retrieval of proteins back to their ‘resident’ compartment.
The major features of membrane trafficking pathways
Secretory/Exocytic (biosynthetic) pathway: ER to Golgi to PM/endosome/lysosome
Endocytic pathway (recycling or degradative): cell surface to endosome to Golgi/ER/lysosome
3 important points about these pathways
Note1.These pathways intersect!!!
Note2. Proteins can be modified (glycosylated and proteolytically cleaved) as they transit the ER and Golgi (important later!)
NOTE: Mitochondria are not part of the secretory pathway
Which compartments form part of the exocytic pathway
Early endosome and lysosome
Recycling endosome and Golgi apparatus
Mitochondria and Endoplasmic reticulum
Endoplasmic reticulum and Golgi apparatus
Endoplasmic reticulum and Golgi apparatus
What did EM and Biochemical analysis show
Em - compartments
Biochem - isolation of compartments ahowing distinct enzymes
How identify components of trafficking
genetic approach - KO gene - identify consequence - deduce function
Which genetic organisms are commonly used?
Mice, Drosophila, fruit flies, C. elegans, S. cerevisiae (budding yeast), etc. can be used, as well as tissue culture cells.
What might make a model suitable for studies on membrane trafficking?
simple with trafficking on cellular scale
More complex if IC trafficking
Yeast as a model organism
Advantages
amenable for genetic studies (can grow as haploid and diploid cells)
entire genome sequence known
cheap and easy to grow in large quantities
limited gene diversity (both ±)
fundamental pathways conserved e.g. cdc
Yeast as a model organism
Disadvantages
limited cell-cell contact so unlikely to be informative about multicellularity
small (5µm), so high resolution imaging studies of intracellular compartments is difficult.
Has a cell wall which can preclude some types of studies
Given the properties of yeast, what type of process is it unlikely to be informative about ? A) Membrane trafficking B) Multicellularity C) Cell cycle regulation D) Cell wall synthesis
A
3 Yeast screens
Sec: secretory
End: endocytic
Vps: vacuole protein sorting
Key experiment by
Novick and Schekman 1980
Key experiment aim
to investigate the secretory pathway in yeast
The experimental analysis of sec pathway
They look for cells which secrete invertase and acid phosphatase, and defined mutants which fail to export the enzymes, but which will synthesize proteins under restrictive growth conditions.
They also looked for cells which ‘look different’ through electron microscopy.
What was found through EM of sec pathway
Secretory mutant cells have a different ultra-structure e.g accumulation of vesicles or aberrant membranous structures
Novick and Schekman experiment rationale
Rationale for their approach - if proteins couldn’t be secreted i.e cells were secretory deficient (sec-), the cell would increase its density as the vesicles carrying the proteins accumulate. They could also look at changes in proteins that are normally secreted.
What genes were identified and what could this say about secretion? Sec
23 genes were identified by grouping mutants with similar phenotypes.
This means that at least 23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membrane.
How were Sec- ordered
Mutant groups were placed in sequential order by combining mutants from different classes and by use of more detailed analysis of protein modifications.
what do certain proteins acquire through sec
Certain proteins show different modifications as they move through the secretory pathway
What happens to alpha factor as moves through sec
Alpha factor is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages this helps us follow its progress
The core protein is glycosylated in the ER. If the protein leaves the ER and travels to the Golgi, it is glycosylated further. Protein stuck in the Golgi will be very big.
In the later parts of the Golgi, the protein is cleaved into smaller pieces, which is secreted as mature alpha factor.
Classification of the sec genes
The 23 genes fell into five classes (A to E), based on the effect on where the protein went and in what way it was modified.
Why weren’t all of the genes/proteins involved in the exocytic pathway identified by Novick and Schekman ?
They only identified temperature sensitive mutants. Not all genes when mutated will cause this phenotype.
They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole would not be identified.
Any ‘redundantly’ functioning genes would not be identified though yeast has relatively low gene redundancy which underpinned the success of these approaches.
Beyond secretion
Further screens were carried for proteins within the lysosome
A decision is made in TGN whether to traffic to surface or towards lysosome.
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