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Genetics of cell growth and division > Hettema - peroxisomes and vacuoles > Flashcards

Hettema - peroxisomes and vacuoles Flashcards

1
Q

three models of biogenesis of cytoplasmic structures?

A
  • Growth and division
  • Templated assembly/growth – structures duplicated e.g. spindle duplication
  • De novo formation
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2
Q

what are the two systems of organelle biogenesis?

A

Endomembrane system: Endoplasmic reticulum and downstream organelles of the secretory pathway
Autonomous organelles: Mitochondria, Chloroplasts (don’t form de novo as they would lose their genome),

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3
Q

Which system do peroisomes belong to?

A

Peroxisomes do not have their own genome but are considered as part of the autonomous system

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4
Q

What are peroxisomes?

A
  • All eukaryotes
  • Single membrane
  • Low abundance
  • Fatty acid ß-oxidation – degradation
  • Several other H202- producing oxidation reactions
  • Catalase degrades H202
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5
Q

what is The old model for peroxisome biogenesis (1985)?

A

Peroxisome grows bigger by protein import and divides when a critical size is reached
This model is more or less correct

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6
Q

What is Zellweger syndrome ?

A 
rare: 1:50.000 
autosomal recessive
Human metabolism and Development are affected 
Most patients die before 1 y old
multigene disorder
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7
Q

How to find the genes involved in peroxisomes?

A
  • Looked at catalase levels as when not localised to peroxisomes is stable in the cytosol
  • Incubated two patient cell lines with PEG which fuses cells
  • Cytoplasm of the cells mix and can be maintained
  • In some, if complementation is occurring, catalase starts to become punctate (dots/peroxisomes)
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8
Q

What is the assay for peroxisome dysfunction in yeast?

A

Yeast can grow of glucose or fatty acids (fatty acid growth requires peroxisomes)
Mutants in peroxisomes cannot grow on fatty acids but can grow of glucose – identified mutants
Either mutants in fatty acid oxidation or peroxisome formation

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9
Q

how many classes of peroxisome biogenesis mutants are there?

A

Class1: defect in matrix protein import e.g PEX1, 2, 4
Class 2: defect in membrane biogenesis e.g PEX3, 19
Class3: Defecs in shape, number and distrubution e.g PEX11, Vps1, DMN1

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10
Q

Most genes identified by homology to yeast PEX genes

A

1) Identify candidate human pex genes
2) Transfect putative PEX genes into patient Fibroblasts and test for complementation
3) Identify mutations in the patient’s copies of the gene that complements.
4) Set up prenatal diagnosis tests

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11
Q

What defects are associated with class 1 mutations?

A

ZSS and RCDP

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12
Q

What defects are associated with class 2 mutations?

A

ZSS

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13
Q

What defects are associated with class 3 mutations?

A

mild ZSS, optic otrophy, CMT2A Leigh like

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14
Q

how do matrix proteins get into the peroxisome?

A
  • PTS1 peroxisomal targeting signal for most peroxisomal enzymes
  • PTS2 peroxisomal targeting signal for some peroxisomal enzymes
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15
Q

How do matrix proteins get into peroxisomes without a PTS?

A
  • Most of these are piggy back imported.
  • Peroxisomal membrane proteins (PMPs) still targeted to membranes
  • Independent of Class1 genes
  • There are 3 routes at least for PMPs.
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16
Q

Model for protein import

A

Newly synthesised protein has a peroxisome targeting signal (PTS1)
Recognised by the receptor Pex5
Complex docks on the membrane by docking complex, Pex13, 14 and 17
Pex 5 becomes/behaves as an integral membrane protein – forms a flexible pore along with Pex14 and probably Pex13 and allows the translocation of folded proteins
Pex5 receptor is ubiquinated and extracted from the membrane
Process involves reversible Ubiquitination

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17
Q

What did GFP-PTS1 live imaging show?

A
  • Multiplication of peroxisomes
  • Retention – some peroxisomes are anchored and some are moving – key for segregation
  • Transport to daughter
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18
Q

What did pulse chase GAL1-GFP-PTS1 show?

A

division is mainly by fission

did not exclude de novo formation

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19
Q

Pulse chase of two differet mating typed showed…

A

that peroxisomes do not fuse

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20
Q

What is the phenotype of a pex3 mutant?

A

no peroxisomes

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21
Q

What did pex3-GFP show ?

A

de novo formation
come from ER
requires pex19

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22
Q

What is the assay for de novo formation?

A
  • Constitutive expression of HcRed-PTS1 – all peroxisomes will be labelled red
  • Pulse GFP-PTS1 (3h) – induced marker, only seen in fusion formed peroxisomes
  • Distinguishes de novo peroxisomes as these will be completely red and will contain no green (looks orange when combined with red)
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23
Q

what were the results of the de novo formation assay?

A

WT cells do not form peroxisomes de novo as well as by fission
Peroxisomes form de novo in cells failing to inherit them
• Inp2 is required for inheritance of peroxisomes
• Inp2 mutants cannot pass on peroxisomes – see de novo peroxisome formation

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24
Q

How do peroxisomes divide?

A

By dynamin-dependent budding

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25
What is dynamin?
* Conserved GTPase * Dynamin is required for scission of vesicles from plasma membrane (PM) * Assembles into oligomeric rings * Self-assembling * Membrane fission events * Signalling GTPase or mechanoenzyme?
26
What are the dynamin-related proteins In S. cerevisiae?
* Vps1p, vacuolar protein sorting * Dnm1, mitochondrial fission * Mgm1, mitochondrial inner membrane fusion/remodelling
27
Vps1 mutant phenotype?
Δvps1 cells have 1-3 peroxisomes per cell – reduction in peroxisome number and funny shaped peroxisomes Vps1 is required for peroxisome number
28
vps1, dnm1 double knockout phenotype
1 peroxisome per cell and elongated structure
29
how is dnm1 recruited to the membrane?
``` by proteins (mdv1, caf1, fis1) knockouts of these give a normal peroxisome phenotype but mitochondrial phenotype affected ```
30
What are the two independent machineries for peroxisome fission?
dnm1, mdv1, caf1, fis1 -> also used for mitochondria Vps1p two pathways may work together
31
What did pulse labelling pex19 mutanst with Pex3-GFP and WT cells with HcRed-PTS1 and mating them show?
Pex3-GFP travels from ER to pre-existing peroxisomes | • The ER provides peroxisomes with essential membrane constituents
32
What affects peroxisome abundance?
• Peroxisome number varies by the cells conditions • Peroxisomes proliferate when needed growth on fatty acids as sole carbon source to compensate for mitochondrial dysfunction • Peroxisomes are actively degraded when no longer required o Switch from oleate back to glucose o Nitrogen starvation
33
What happens in peroxisome proliferation?
* Induction of beta-oxidation enzymes – only when no glucose * Induction of a few genes required for peroxisome biogenesis (including Pex11) * Increase in peroxisome number and size * This must be a tightly regulated process as it is a large investment for the cell
34
What is the oleate response?
• Key transcriptional activator Oaf1/Pip2 heterodimer Fatty acid binds Oaf1p, oaf1 binds pip2p • Recognises oleate response elements in promoters. • Acts via a asymmetric positive feedback loop – control
35
What is the asymmetric feedback loop?
1. A heterodimer forms in response to a signal to regulate activity of downstream target 2. One component of the heterodimer acts as a sensor of the signal, and one component is also target of feedback regulation by the heterodimer itself. 3. This motif confers precise, tunable and robust control of responses to environmental stimuli
36
Features of the feedback loop?
System has a slow induction Very sensitive to transcription factor concentration System plateaus
37
How do peroxisomes segregate during cell division?
Get retention of certain peroxisomes in mother cell and transport of others G1: Peroxisomes are initially segregated throughout the cell S: Peroxisomes move to the bud – sometimes before the bud is even visible G2: Seen in cell tip and others stay in mother G1: move back to site of cytokinesis
38
what does disabelling actin filaments do?
stops peroxisome movement
39
What does myo2 CBD overexpression inhibit?
segregation as peroxisomes can't bind
40
Myosin
Myosin is a multi-domain protein with an ATPase domain, a coiled coil domain and a cargo binding domain (CBD)
41
how do peroxisomes segregate?
* Transport along actin cables * Myo2p is the motor protein * Inp1 Binds peroxisome to Myo2
42
What does Inp1 do in peroxisome segregation?
Inp1p anchors the peroxisome to the cortex of the mother cell Peroxisomes are retained in mother cell by INP1 Inp1 knockout has peroxisomes localised in the bud Inp1 overexpression has all peroxisomes in the mother
43
What does pex3 do in peroxisome segregation?
Pex3 anchors Inp1 to peroxisomes
44
what are the two functions of pex3?
1. Peroxisome formation from ER | 2. Peroxisome retention
45
Model peroxisome segregation and multiplication
Cells are retained in the mother by anchoring at the periphery of the cell Myosins are pulling on the peroxisomes transporting them to the bud Dynamin-related proteins pinch off the peroxisomes
46
KO and DAMP screen
to identify factors required for growth of peroxisomes | identified triple knockouts
47
What in Inp3?
A kinase - Kin4 partially redundant with its paralogue Frk1 double knockout gives clear phenotype Mitochondrial inheritance is not blocked
48
Synthetic genetic array technology
1. Mate query strain (selectable marker) with library of known mutants containing the kanR gene arrayed in 384 well plates 2. Select diploids 3. Sporulate 4. Select Mat alpha haploids 5. Select kanR 6. Select kanR and other selectable marker
49
Does vacuole inheritance depend on actin and Myo2
yes
50
what is the vac8/17 mutant phenotype?
blocked inheritance
51
when is vac 17 broken down?
upon entry to bud
52
How does vacuole inheritance occur?
``` cdc28 phosphorylates vac17 Vac8 and Vac17 form complex on vacuolar membrane that recruits Myo2 (Vac17 binding is via Vac8) Vac17 interacts with CBD on Myo2 vacuole dragged to bud segregation structure falls apart vacuole is deposited Vac17 is degraded ```
53
what is myo2 release from cargo regulated by?
Dma1
54
What is Dma1?
ubiquitin ligase | required for Vac17 degradation
55
What does Dma1 do?
recognises Vac17 FTA domain recognises phosphothreonine residues Binds Vac17 at PEST sequence causes vac17 degradation
56
What residues does Dma1 recognise?
T240 | S222
57
What does inactiation of the PAK kinases cause?
1) Stabilises Vac17 Kinases normally keep the level of Vac17 low – inactivation means they are no longer breaking Vac17 down 2) Repositions vacuoles to budneck Close to the site of cytokinesis as they are still attached to the actin cables
58
What does Cla4 do?
Phosphorylates Vac17 at s222 it is a cdc42 effector recruited to sites of polarised growth
59
How does Cla4 causes dma1 to ubiquitinate Vac17?
Either: Binding partner activates Dma1 Vac17 undergoes a conformational change
60
What is Kin4 required for?
Vacuole inheritance It is a mother cell-specific kinase Regulates spindle position check point Kin4 is active in the mother – mitosis is inhibited Kin4 enters the bud and is inactivated – mitosis can occur
61
What acivates Kin4?
Elm1
62
What inhibits Kin4?
Cla4
63
What does overexpression of Kin4 and Frk1 cause?
increased Vac17 levels and vacuole close to site of cytokinesis
64
How do all cells have vacuoles?
Inherited | if not inherited they are formed de novo
65
What are Kin4 and Frk1 required for?
vacuole transport to bud | protect Vac17 from breakdown in the mother
66
What is the phenotype of a Kin4/Frk1 double knockout
Vac17 levels are decreased
67
How are peroxisomes moved?
Like vacuoles | Myo2 interaction mediated by Inp2
68
What is the phenotype of a Frk/kin4 double knockout in peroxisomes?
Inheritance defects | decreased inp2
69
What does overexpression of Cla4 in peroxisomes cause?
inheritance defect (none in bud)
70
What happens in a dma1/dma2 double knockout in peroxisomes?
accumulation of Vac17 and inp2
71
regulators of vac stability also affect?
inp2
72
What happens in a dma1/dma2 double knockout in mitochondria?
increase receptor levels in mitochondrial dynamics
73
What is the phenotype of a Frk/kin4 double knockout in mitochondria?
No inheritance defects | must have additional regulating factors

Genetics of cell growth and division (4 decks)

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