Using yeast to understand membrane trafficking

Question 1

why do eukaryotic cells need membrane trafficking?

Answer 1

• for sequential modifications, proteins need to be exposed to different enzymes for processes such as glycosylation or proteolytic cleavage
• membrane trafficking is important to expose the proteins to these enzymes step-by-step to allow specific order of modifications
• membrane trafficking also allows retrieval or proteins back to their resident compartment

Question 2

what are the main features of the secretory/exocytic (biosynthetic) pathway?

Answer 2

ER -> Golgi -> plasma membrane/endosome/lysosome
- during protein synthesis, proteins are translated to the ER lumen and trafficked to the Golgi
- proteins then move through the Golgi into the trans-Golgi network (TGN) for sorting
- proteins are then constitutively secreted or regulatory secreted from the TGN into secretory vesicles

Question 3

what are the main features of the endocytic pathway (recycling or degradative)?

Answer 3

cell surface -> endosome -> Golgi/ER/lysosome
- can recycle material from cell surface
- can degrade material if the cell already has enough of a certain substrate
- enables transport of a receptor from one part of the plasma membrane to another area

Question 4

where can the secretory and endocytic pathways intersect?

Answer 4

at the lysosome

Question 5

what happens to proteins as they travel through the secretory or endocytic pathways?

Answer 5

proteins can be modified as they transit the ER and Golgi
- they can be glycosylated by addition of oligosaccharides
- they can be proteolytically cleaved

Question 6

how are proteins modified by oligosaccharide addition in the ER and Golgi?

Answer 6

Both N- and O- linked glycosylation can occur on proteins:
1. In the ER lumen, addition of pre-formed oligosaccharide to an asparagine amino acid in a consensus sequence (followed by Ser or Thr)
2. addition and processing of oligosaccharides during transit of secretory pathway
3. in the Golgi, the oligosaccharide group is trimmed
4. Later in the Golgi, further sugars are added and the structure can be branched by sequential modifications
- recognition targets for pathogens

Question 7

why is sugar addition useful to proteins?

Answer 7

• sugars are extremely heavy (100kDa), so can determine the folding of a protein

Question 8

what happens if sugar modification goes wrong/doesn’t happen?

Answer 8

if sugar modification doesn’t occur, the protein may not fold correctly and so will be unable to bind to receptors

Question 9

what is the purpose of glycosylation?

Answer 9

1. to assist folding
2. as a ligand for:
   - intracellular for trafficking/sorting
   - extracellular for interactions with ECM and proteins/sugars on other cells

Question 10

does glycosylation occur in the same way to all proteins?

Answer 10

no, not all proteins will have oligosaccharides modified in the same way

Question 11

what genetic organisms were commonly used to identify the genes involved in trafficking pathways?

Answer 11

• Mouse, Drosophila melanogaster (Fruit fly), Zebrafish, Caenorhabditis elegans (nematode), Dictyostelium discoideum, Saccharomyces cerevisiae (budding yeast).
• Can also use tissue culture cells for studies.

Question 12

what makes a model suitable for studies o membrane trafficking?

Answer 12

• Simplicity - trafficking occurs on a cellular scale so a single-celled organism is likely to provide information.
• Analysis of specific types of secretion e.g. regulated secretion, would need a model system that is able to perform this function.
• Budding yeast does not do regulated secretion, but they can be modified genetically to do this
• Often different systems reveal different information.

Question 13

what are the advantages of Saccharomyces cerevisiae (budding yeast) as a model organism for membrane trafficking?

Answer 13

• 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 (good as there is little redundancy)
• fundamental pathways conserved

Question 14

what are the disadvantages of Saccharomyces cerevisiae (budding yeast) as a model organism for membrane trafficking?

Answer 14

• limited cell-cell contact so unlikely to be informative about multicellularity signals
• small (5µm), so high resolution imaging studies of intracellular compartments is difficult.
• Has a cell wall which can preclude some types of studies

Question 15

what were the 3 main yeast screens used to study membrane trafficking?

Answer 15

1. sec = secretory pathway
2. end = endocytic pathway
3. vps = vacuolar protein sorting

Question 16

what experiment did Novick and Scheckman conduct to study the sec secretory pathway?

Answer 16

hypothesis: if proteins couldn’t be secreted (cells were secretory deficient - sec-), the cell would increase in density due to vesicle accumulation

1. cells were analysed for their ability to secrete enzymes at permissive and high temps
   - they defined sec mutants as those strains which failed to export invertase and acid phosphatase, but continued to make protein under restrictive growth conditions - global defect in secretion
2. observed alterations in cell ultra-structure using EM e.g. accumulation of vesicles or aberrant membranous structures
   - they mutagenised the cells, took the heaviest ones and analysed them under EM

Question 17

how do sec mutant cell structure differ with wild type yeast cells?

Answer 17

normal yeast cell has a bud and contains organelles

sec mutant yeast cell contains accumulation of vesicles and membranes due to the lack of secretory enzymes

Question 18

what did the sec experiments by Novick and Schekman help to discover?

Answer 18

• 23 sec genes were identified by grouping mutants with similar phenotypes
• at least 23 distinct gene products are required to ensure the transport of proteins from the ER to the PM via the secretory pathway
• mutant groups were placed in sequential order by combining mutants from different classes and by detailed analysis of protein modifications

Question 19

what are the 5 sec gene classes?

Answer 19

1. Class A = protein couldn’t enter ER, so these genes important for ER uptake
2. Class B = couldn’t leave ER so important in budding of vesicles from ER
3. Class C = proteins found trapped in vesicles, so these are important for fusion with Golgi
4. Class D: proteins entered Golgi but was trapped so these important for leaving Golgi
5. Class E: vesicles trapped at surface, so important for docking at plasma membrane

Question 20

why weren’t all of the genes involved in the secretory pathway identified by Novick and Schekman?

Answer 20

1. They only identified temperature-sensitive mutants. Not all genes when mutated will cause this phenotype.
2. They only considered secretion to the plasma membrane so defects in transport to endosome or vacuole would not be identified.
3. Any ‘redundantly’ functioning genes would not be identified though yeast has relatively low gene redundancy which underpinned the success of these approaches.

Question 21

what modifications do proteins show as they move through the secretory pathway?

Answer 21

• Alpha factor is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages this helps us follow its progress
• When moved to Golgi, many sugars are added
• In late golgi, just before secretion, it is proteolytically cleaved into small peptides for secretion

Can trace these stages to see where modifications are made using blotting

Question 22

what occurs in the trans-Golgi network?

Answer 22

A decision is made in TGN whether to traffic to cell surface or towards lysosome for degradation

Question 23

what is endocytosis?

Answer 23

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

Question 24

why is endocytosis important?

Answer 24

1. Retrieval of molecules that formed part of the secretory vesicle for recycling
2. Downregulation of signals
3. Remodeling cell surface lipid and protein composition under different environmental strains

Note: endocytosis is also a means of entry into cells for many pathogens and toxins

Question 25

what are the 4 main stages of the endocytic pathway?

Answer 25

1. Plasma membrane invaginates formed endocytic vesicle
2. Endocytic vesicle to early endosome
3. Early endosome to late endosome (MVB) or recycling to the plasma membrane
4. Late endosome to Golgi or vacuole for degradation

Question 26

what did the end- screens in yeast help discover about endocytosis?

Answer 26

• End- screens looked for mutants that could not internalise a fluid phase marker (lucifer yellow) or a bound pheromone alpha-factor.
• 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 invagination

Question 27

what is the major function of the lysosome/vacuole?

Answer 27

the degradation of extracellular material taken up by endocytosis as well as certain intracellular components by a process termed autophagy

Question 28

what is autophagy?

Answer 28

Autophagy = degradation of organelles

Question 29

why is lysosome compartmentalisation important?

Answer 29

these organelles contain many degradative/proteolytic enzymes that must be kept separate from the rest of the cell, as they may cause cellular damage

Question 30

how do lysosomes receive their degradative enzymes?

Answer 30

• The lysosome’s resident enzymes are transported to the lysosome through the secretory pathway.
• At the late Golgi compartment (Trans Golgi Network), they are sorted into a pathway destined for lysosomes rather than the plasma membrane.
• Genetic studies in yeast were again central to the identification of genes/proteins involved in this sorting and trafficking pathway.

Question 31

what were the vacuolar protein sorting (vps) screens?

Answer 31

• 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 secreting CPY were investigated further using microscope-based and biochemical techniques.
• Again many genes were identified and these fell into distinct groups.
• Over 60 vacuolar protein sorting (vps) genes have been identified in this way.
• 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

Question 32

what is CPY?

Answer 32

Carboxypeptidase Y (CPY) is normally transported to the lysosome having been trafficked through the ER and Golgi
- it is a degradative enzyme which was studied in vps screens

Question 33

how was CPY studied under biochemical analysis in the vps screens?

Answer 33

• As with alpha-factor, CPY is glycosylated (has long sugar chains added) and proteolytically cleaved at different stages. This helps us follow its progress
• At vacuole, the pre-sequence of CPY which regulates its function is cleaved to form active CPY

Question 34

how was CPY analysed using fluorescence microscopy in the vps screens?

Answer 34

using a fluid phase dye – lucifer yellow - helped to identify different morphological features of cells

Question 35

How are vacuolar mutants divided into classes?

Answer 35

They are divided into classes depending on the stage at which they appear to block the route to the vacuole
- set of proteins were detected that couldn’t leave the TGN
- some proteins that couldn’t enter the early or late endosomes were detected - showed more than one route between TGN and late endosome
- there was accumulation of proteins at MVBs - issue with final fusion step

Question 36

what are Multivesicular bodies (MVBs)?

Answer 36

these fuse with the lysosome to deposit degradative proteins

Question 37

what are the 4 possible trafficking pathways/destinations from the late Golgi/TGN?

Answer 37

1. To plasma membrane - sec
2. To early endosome - end
3. To late endosome/MVB - CPY
4. To vacuole - ALP

Question 38

How are proteins sorted into the late endosome/MVB (CPY pathway)?

Answer 38

1. CPY is synthesized in a prepro form (p2CPY) and is transported through the ER to the Golgi.
2. Sorting: in the late Golgi CPY is specifically recognised by a receptor Vps10 i.e. sorting is receptor-mediated.
3. The transport step requires cytoplasmic factors: Clathrin and two adaptors called Gga1 and Gga2.
4. CPY dissociates from Vps10 at the late endosome/MVB and is transported to vacuoles where it is cleaved to generate the mature form.
5. Vps10 is retrieved to the late Golgi through a specific aromatic-based signal which is received by an adaptor protein in its protein sequence (YSSL, FYVF)