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.
.
Endocytosis define
Endocytosis is the process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that is then able to fuse with endosomes and enter the endo-lysosomal membrane system.
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
Note: endocytosis is also a means of entry into cells for many pathogens and toxins
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
What did endocytosis screens look at
End- screens looked for mutants that could not internalise a fluid phase marker (lucifer yellow) or a bound pheromone alpha-factor.
Result end- screen
7 end- genes detected. Of these 5 directly involved in the process of membrane invagination and scission – identification indicated importance of actin cytoskeleton in yeast endocytosis
Vacuolar/Lysosomal protein sorting function
The 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.
Vacuoles and lysosomes contain
Thus, these organelles contain many degradative/proteolytic enzymes that must be kept separate from the rest of the cell.
The lysosome’s resident enzymes are transported to
lysosome through the secretory pathway.
At the late Golgi compartment (Trans Golgi Network) what occurs
they are sorted into a pathway destined for lysosomes rather than the plasma membrane
The Vacuolar protein sorting (vps) screens: what should happen
Carboxypeptidase Y (CPY) is normally transported to the lysosome having been trafficked through the ER and Golgi.
What happened in Vps-
Several labs generated mutagenised cells and looked for cells which secreted CPY (using a simple colour based assay) which is normally a vacuolar enzyme.
Cells which secreted CPY were then investigated further using microscope based and biochemical techniques.
How many Vps genes identified
Over 60 vacuolar protein sorting (vps) genes
How were Vps genes ordered
Just as the sec mutants were combined to determine the order of action of the genes - so this was done for the vps mutant strains.
Analysis of biochemical analysis of Vps showed
As with a-factor CPY is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages this helps us follow its progress
The protein carboxypeptidase Y (CPY) has been used in yeast genetic screens to allow identification of distinct trafficking compartments. What properties of CPY are central to its use in the screens
It is ubiquitylated on its cytoplasmic face for recognition by adaptor proteins
It is extensively mannosylated in the Golgi apparatus
It is phosphorylated in early endosome
It is glycosylated and proteolytically processed in different compartments
D
How are vacuolar mutants divided into classes
Vacuolar mutants are divided into classes depending on the stage at which they appear to block the route to the vacuole
Draw out pathway
See notes
Trafficking pathways from the late Golgi – 4 poss destinatons
- To plasma membrane
- To early endosome
- To late endosome/MVB
- To vacuole
Trafficking pathways from the late Golgi – 4 poss destinatons –> HOW DO THEY KNOW
There are molecular determinants - signals on the proteins or on sugars - that can signal to other proteins where a particular protein needs to go: the plasma membrane, the early endosome, the late endosome, or the vacuole.
Sorting to the Late Endosome/MVB - The CPY pathway
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).
