Yeast membrane trafficking Flashcards
Why do eukaryotic cells need membrane trafficking? (4)
- Compartmentalisation allows more complexity
- Enzymes can modify specific subsets of proteins in certain compartments
- Proteins need to be exposed to distinct sets of enzymes for sequential modifications
- Needed to retrieve proteins back to their ‘resident’ compartment
What are the 2 major features of membrane trafficking pathways?
- Secretory/exocytic (biosynthetic)
- Endocytic (recycling/degradative)
What is the secretory/exocytic pathway? (4)
- Proteins enter the ER lumen during synthesis
- Trafficked to the Golgi, ends up in the trans-Golgi network
- Proteins secreted via constitutive secretion/regulated secretion
- Enzymes being made go through the pathway and are trafficked to lysosomes via transport vesicles
What is the trans-Golgi network?
Sorting compartment of the Golgi apparatus
What is constitutive secretion?
Secretion of proteins without specific signals on them
What is regulated secretion?
Proteins sorted into secretory vesicles and held there until a signal is received for secretion
What is the endocytic pathway? (2)
- Cell surface to the late Golgi/ER/lysosome
- Intersects with the exocytic pathway
How are proteins modified during trafficking?
Proteins can be glycosylated/proteolytically cleaved as they transit the ER and Golgi
What is glycosylation?
Addition of oligosaccharides
What are the 2 types of glycosylation that can occur during exocytosis
- N-linked glycosylation
- O-linked glycosylation
What is N-linked glycosylation?
Attachment of oligosaccharides to a nitrogen atom within asparagine
What is O-linked glycosylation?
Attachment of oligosaccharides to an oxygen atom in serine or threonine
What is the process of glycosylation? (5)
- Pre-formed large oligosaccharide is attached to asparagine/serine/threonine site when the protein is being made on the rER membrane
- The oligosaccharide is trimmed down at the ER
- The protein is moved to the Golgi in a vesicle, sugar trimmed down again
- Further sugars are added as the protein moves through the Golgi
- The sugars at the ends of branches are often important for recognition
What is the purpose of glycosylation? (2)
- To assist folding
- As a ligand: intracellular for trafficking/sorting, extracellular for interactions with ECM/proteins/sugars on other cells
What are the advantages of yeast as a model organism? (4)
- Amenable for genetic studies (can be haploid and diploid cells)
- Entire genome sequence is known
- Limited gene diversity
- Fundamental pathways are conserved
What are the disadvantages of yeast as a model organism? (3)
- Limited cell-cell contact so not useful for multicellularity information
- Small so high resolution imaging of intracellular compartments is difficult
- Has a cell wall so can’t microinject
What are the steps of secretion in yeast? (4)
- ER
- ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network
- Cisternal progression
- Constitutive secretion
What are the 3 main yeast screens that were done to understand membrane trafficking?
- Sec (secretion) screen
- End (endocytosis) screen
- Vps (vacuole protein sorting) screen
Who performed the sec screen?
Novick and Schekman
What was the rationale behind Novick and Schekman’s sec screen experiment?(2)
- If proteins couldn’t be secreted (i.e. cells were secretory deficient, sec-), the cell would increase in density because the vesicles would accumulate
- Also looked at changes in proteins that are normally secreted
Which enzymes were investigated in the sec screen? (2)
- Invertase
- Acid phosphatase
What was the experimental analysis in the sec screen? (3)
- Mutant cells were analysed for their ability to secrete enzymes at permissive and restrictive temperatures
- Cells which failed to export active invertase and acid phosphatase were defined as secretory mutants
- Electron microscopy was used to observe abnormal ultrastructure e.g. accumulation of vesicles
How did the ultrastructure of secretory mutant cells differ from wild-type?
Sec- mutants showed accumulation of vesicles when observed by electron microscopy
What was the result of the sec screen?
23 sec genes were identified by grouping mutants with similar phenotypes
What groups were sec mutants separated into? (5)
- Protein accumulation in the cytosol (couldn’t get into the ER)
- Accumulation in rough ER (couldn’t bud from the ER)
- Accumulation in ER-to-Golgi transport vesicles (vesicles couldn’t fuse with the Golgi)
- Accumulation in the Golgi (couldn’t get into secretory vesicles from the Golgi)
- Accumulation in secretory vesicles (couldn’t transport from secretory vesicles to the cell surface)
Why weren’t all of the genes involved in the exocytic pathway identified by Novick and Schekman in the sec screen? (3)
- They only identified temperature sensitive mutants, not all mutated genes show this phenotype
- They only investigated secretion to the plasma membrane so defects in transport to the endosome/vacuole weren’t identified
- Any redundant genes couldn’t be identified
How was alpha-factor used to find out the stages of the secretory pathway? (4)
- Alpha-factor is glycosylated in the ER
- Lots more sugars are added in the Golgi
- Alpha-factor is proteolytically cleaved before secretion
- The modification stages were used to identify mutants with defects in the stages of the pathway, not just overall secretion from the plasma membrane
What is endocytosis?
The process through which the plasma membrane invaginates into the cell resulting in the production of a vesicle that fuses with endosomes and enters the endo-lysosomal membrane system
Why is endocytosis important? (4)
- Retrieval of molecules that form the secretory vesicle to deliver cargo to the membrane so they can be recycled
- Getting rid of membrane receptors for downregulation of signals
- Remodelling cell surface lipid and protein composition
- Also a method of entry for pathogens and and toxins
What are the stages in the endocytic pathway in yeast? (4)
- Invagination of the plasma membrane to form an 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 is the MVB? (2)
- Multi Vesicular Body (late endosome)
- Fuses with the lysosome to deposit its contents to be degraded
What is the mammalian functional equivalent of the yeast vacuole?
Mammalian lysosome
How was the end screen performed?
End- screens looked for mutants that couldn’t internalise a fluid phase marker
What fluid phase marker was used in the end- screens?
Lucifer yellow
What were the results of the end screen? (2)
- 7 genes were identified, 5 of which are directly involved in membrane invagination and scission
- Identification of the importance of the actin cytoskeleton in yeast endocytosis
What is the function of the lysosome (vacuole)?
Degradation of extracellular material taken up by endocytosis as well as intracellular components by autophagy
What is the purpose of the vps (vacuolar/lysosomal protein sorting) pathway? (2)
- Lysosomal enzymes for degradation need to be kept separate from the rest of the cell
- Therefore they are sorted at the trans Golgi network into a pathway destined for lysosomes rather than the plasma membrane
What is CPY? (2)
- Carboxypeptidase Y
- Transported to the lysosome/vacuole after being trafficked through the ER and Golgi
How were the vps screens done? (4)
- Identified mutants which secreted CPY instead of it being sent to the vacuole
- Analysed the mutants microscopically and biochemically
- Identified over 60 vps genes
- CPY is modified along the pathway and cleaved when it enters the lysosome to become active
What are the 4 trafficking pathways from the late Golgi?
- To the plasma membrane
- To the early endosome
- To the late endosome/MVB (CPY pathway)
- To the vacuole (ALP pathway)
How is CPY sorted into the late endosome? (4)
- p2 form of CPY in the Golgi is recognised by Vps10
- CPY-bound Vps10 is recognised by cytoplasmic factors and sorted into a vesicle which travels to the late endosome
- CPY dissociates from Vps10 in the late endosome/MVB and is transported to the vacuole where it is cleaved into the active form
- Vps10 is retrieved from the late endosome and sorted into vesicles by adaptor proteins
