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L2 Molecular Cell Biology > Yeast membrane trafficking > Flashcards

Yeast membrane trafficking Flashcards

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1
Q

Why do eukaryotic cells need membrane trafficking? (4)

A
  • 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
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2
Q

What are the 2 major features of membrane trafficking pathways?

A
  • Secretory/exocytic (biosynthetic)
  • Endocytic (recycling/degradative)
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3
Q

What is the secretory/exocytic pathway? (4)

A
  • 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
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4
Q

What is the trans-Golgi network?

A

Sorting compartment of the Golgi apparatus

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5
Q

What is constitutive secretion?

A

Secretion of proteins without specific signals on them

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6
Q

What is regulated secretion?

A

Proteins sorted into secretory vesicles and held there until a signal is received for secretion

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7
Q

What is the endocytic pathway? (2)

A
  • Cell surface to the late Golgi/ER/lysosome
  • Intersects with the exocytic pathway
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8
Q

How are proteins modified during trafficking?

A

Proteins can be glycosylated/proteolytically cleaved as they transit the ER and Golgi

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9
Q

What is glycosylation?

A

Addition of oligosaccharides

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10
Q

What are the 2 types of glycosylation that can occur during exocytosis

A
  • N-linked glycosylation
  • O-linked glycosylation
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11
Q

What is N-linked glycosylation?

A

Attachment of oligosaccharides to a nitrogen atom within asparagine

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12
Q

What is O-linked glycosylation?

A

Attachment of oligosaccharides to an oxygen atom in serine or threonine

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13
Q

What is the process of glycosylation? (5)

A
  • 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
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14
Q

What is the purpose of glycosylation? (2)

A
  • To assist folding
  • As a ligand: intracellular for trafficking/sorting, extracellular for interactions with ECM/proteins/sugars on other cells
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15
Q

What are the advantages of yeast as a model organism? (4)

A
  • Amenable for genetic studies (can be haploid and diploid cells)
  • Entire genome sequence is known
  • Limited gene diversity
  • Fundamental pathways are conserved
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16
Q

What are the disadvantages of yeast as a model organism? (3)

A
  • 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
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17
Q

What are the steps of secretion in yeast? (4)

A
  • ER
  • ER-Golgi intermediate compartment (ERGIC)/cis-Golgi network
  • Cisternal progression
  • Constitutive secretion
18
Q

What are the 3 main yeast screens that were done to understand membrane trafficking?

A
  • Sec (secretion) screen
  • End (endocytosis) screen
  • Vps (vacuole protein sorting) screen
19
Q

Who performed the sec screen?

A

Novick and Schekman

20
Q

What was the rationale behind Novick and Schekman’s sec screen experiment?(2)

A
  • 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
21
Q

Which enzymes were investigated in the sec screen? (2)

A
  • Invertase
  • Acid phosphatase
22
Q

What was the experimental analysis in the sec screen? (3)

A
  • 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
23
Q

How did the ultrastructure of secretory mutant cells differ from wild-type?

A

Sec- mutants showed accumulation of vesicles when observed by electron microscopy

24
Q

What was the result of the sec screen?

A

23 sec genes were identified by grouping mutants with similar phenotypes

25
Q

What groups were sec mutants separated into? (5)

A
  • 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)
26
Q

Why weren’t all of the genes involved in the exocytic pathway identified by Novick and Schekman in the sec screen? (3)

A
  • 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
27
Q

How was alpha-factor used to find out the stages of the secretory pathway? (4)

A
  • 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
28
Q

What is endocytosis?

A

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

29
Q

Why is endocytosis important? (4)

A
  • 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
30
Q

What are the stages in the endocytic pathway in yeast? (4)

A
  • 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
31
Q

What is the MVB? (2)

A
  • Multi Vesicular Body (late endosome)
  • Fuses with the lysosome to deposit its contents to be degraded
32
Q

What is the mammalian functional equivalent of the yeast vacuole?

A

Mammalian lysosome

33
Q

How was the end screen performed?

A

End- screens looked for mutants that couldn’t internalise a fluid phase marker

34
Q

What fluid phase marker was used in the end- screens?

A

Lucifer yellow

35
Q

What were the results of the end screen? (2)

A
  • 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
36
Q

What is the function of the lysosome (vacuole)?

A

Degradation of extracellular material taken up by endocytosis as well as intracellular components by autophagy

37
Q

What is the purpose of the vps (vacuolar/lysosomal protein sorting) pathway? (2)

A
  • 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
38
Q

What is CPY? (2)

A
  • Carboxypeptidase Y
  • Transported to the lysosome/vacuole after being trafficked through the ER and Golgi
39
Q

How were the vps screens done? (4)

A
  • 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
40
Q

What are the 4 trafficking pathways from the late Golgi?

A
  • To the plasma membrane
  • To the early endosome
  • To the late endosome/MVB (CPY pathway)
  • To the vacuole (ALP pathway)
41
Q

How is CPY sorted into the late endosome? (4)

A
  • 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

L2 Molecular Cell Biology (13 decks)

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