yeast screens Flashcards
Why do eukaryotic cells need membrane trafficking?
¥ Compartmentalization 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.
Membrane trafficking is also important in 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
Yeast as a model organism
Advantages:
amenable for genetic studies (can grow as haploid and diploid cells)
entire genome sequence known since 1996 (and is fully annotated),cheap and easy to grow in large quantities (good for biochemical studies),
limited gene diversity (both ±)
fundamental pathways conserved
Yeast as a model organism
disadvantages
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
Novick and Schekman 1980 experiments
what was the aim of their experiments
Aim: to investigate the secretory pathway in yeast.
Novick and Schekman 1980
what enzymes were studied in the Sec- analysis done
cells analysed for their ability to secrete enzymes invertase and acid phosphatase
what are secretory mutants
secretory mutants as those strains which fail to export active invertase and acid phosphatase, but continued to synthesize protein under restrictive growth conditions
what was seen in sec mutants
1 alterations in the normal ultra-structure of cells could be observed e.g accumulation of vesicles or aberrant membranous structures. Secretory mutant cells have a different ultra-structure
How many sec genes were identified
23 distinct gene products are required to ensure the transport of proteins from the ER to the plasma membran
how did they split the Sec- genes into the 5 classes?
Mutant groups were placed in sequential order by combining mutants from different classes and by use of more detailed analysis of protein modifications
Class A - fate of secreted proteins
accumulate in cytosol as cannot be transported into the ER
Class B - fate of secreted proteins
accumulate in rough ER as budding of vesicles from ER doesn’t occur
Class C - fate of secreted proteins
accumulation i ER-to-Golgi transport vesicles as no fusion of vesicles to golgi
Class D - fate of secreted proteins
accumulation in golgi as no transport from golgi to secretory vesicles
Class E - fate of secreted proteins
Accumulation in secretory vesicles as transport to cell surface does not occur
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.
what do Sec screens show
mutations affecting ER-plasma membrane but proteins are also synthesized and need to be trafficked to the lysosome. A decision is made in TGN whether to traffic to surface or towards lysosome.
What is Endocytosis ?
process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle
Why is endocytosis important
- Retrieval of molecules that formed part of the secretory vesicle for recycling
- Downregulation of signals
- Remodelling cell surface lipid and protein composition
Stages in the endocytic pathway
Plasma membrane to endocytic vesicle
Endocytic vesicle to early endosome
Early endosome to late endosome (MVB) or recycling to the plasma membrane
Late endosome to Golgi or vacuole
End- screens looked for … and what did it show the importance of
mutants that could not internalise a fluid phase marker (lucifer yellow) or a bound pheromone alpha-factor.
identification indicated importance of actin cytoskeleton in yeast endocytosis
major function of the lysosome (vacuole
is the degradation of extracellular material taken up by endocytosis as well as certain intracellular components by a process termed autophagy
Carboxypeptidase Y (CPY) normal transport route
ER - golgi - lysosome
Carboxypeptidase Y (CPY) how is it modified at different stages and what receptors or cytoplasm factors are required
- CPY is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages this helps us follow its progress
- CPY is synthesized in a prepro form and is transported through the ER to the Golgi.
- Sorting: in the late Golgi CPY is specifically recognised by a receptor Vps10 i.e sorting is receptor-mediated.
- The transport step requires cytoplasmic factors: clathrin and two adaptors called Gga1 and Gga2.
- CPY dissociates from Vps10 at the late endosome/MVB and is transported to vacuoles where it is cleaved to generate the mature form.
- Vps10 is retrieved to the late Golgi through a specific aromatic -based signal in its protein sequence (YSSL, FYVF).
