Lecture 14: Lysosomes, peroxisomes
and mitochondria
Lysosomes, peroxisomes, mitochondria
1. Lysosome structure and function
2. Lysosome targeting
3. Autophagy
4. Peroxisome structure and function
5. Mitochondrial evolution, structure and function
6. Mitochondrial quality control
Lysosomes
Features of Lysosomes
• Heterogeneous appearance
• Range in size from 25nm to 1µm in diameter
• Contain acid hydrolases (active at pH 4)
• High internal proton concentration is maintained by a
proton transporter (H+- ATPase) on the lysosomal
membrane.
• Degrades phagocytosed extracellular material and
autophagosed intracellular material
After internalization, endocytosed cargo is
sorted in endosomes
Figure 13-53 Molecular Biology of the Cell (© Garland Science 2008)
Lysosomes degrade phagocytosed
materials
Phagocytosed axon debris is degraded in lysosomes
Lamp1-GFP
Jeff Rasmussen
Targeting of Lysosomal Enzymes to Lysosomes
Lysosomes also degrade intracellular
materials, such as damaged mitochondria, by
a process called autophagy
Autophagasomes have two membranes
Peroxisomes
Membrane-bound organelles of 0.1 to 1µm in diameter that
contain a dense, crystalline core of oxidative enzymes.
Purified peroxisomes isolated
by centrifugation through a
sucrose density gradient
Functions of Peroxisomes
1. Synthesis and degradation of hydrogen peroxide
2. Oxidation of long-chain fatty acids (fatty acids
with 24 to 26 carbons)
3. Synthesis of cholesterol and bile acids in the liver
4. Synthesis of certain phospholipids in neurons
Peroxisome-resident proteins are synthesized in the cytoplasm
and translocated into peroxisomes
Pex mutants in yeast have defects in peroxisome biogenesis
--Pex mutants were isolated based on their inability to grow on oleic acid
--In Pex mutants peroxisomal matrix proteins accumulate in cytosplasm
--No peroxisomes are evident by EM
WT
Pex1
Erdmann R, Veenhuis M, Mertens D, Kunau WH. (1989). Isolation of peroxisome-deficient mutants
of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 86(14):5419-23.
Proteins defective in Pex mutants are required at different steps of
peroxisomal biogenesis
Pex3 and Pex19
are required for exit
from the ER
Pex1 and Pex6 are
required for
maturation of
preperoxisomal
vesicles
Pex2, 10, 12, and 13 are
required for import of
matrix proteins
Mitochondria
Mitochondria are distributed by
microtubule-based transport throughout
eukaryotic cells
Mitochondrial Structure
Contains two
membranes--inner and
outer; inner membrane
folds into cristae
Two compartments:
1. Intermembrane space
2. Matrix (contains high
concentration of
proteins, DNA,
ribosomes)
Dimensions:
0.2 - 1um diameter
1 - 4 um length
Mitochondrial Membranes
Inner and outer membranes contain distinct proteins and
proteins must be imported through dedicated complexes
Mitochondrial Function
Oxidative metabolism: conversion of products from the
catabolism of carbohydrates, fat and protein into chemical
energy stored in ATP
Steps:
1. Conversion of pyruvate to Acetyl CoA
2. Tricarboxylic Acid (TCA) cycle (Krebs cycle)
3. Electron transport chain
4. Generation of ATP by ATP synthase
How is glucose transformed into energy?
Electron Transport Chain
Electron transport and generation of ATP
ATP
synthase
Mitochondrial quality control
Electron transport generates reactive oxygen species,
which can damage DNA and oxidize proteins
How does a cell deal with damaged or aged mitochondria?
1. Repair: Fission and fusion
2. Send vesicles with damaged components to
peroxisomes and lysosomes
3. Destroy mitochondria through autophagy (mitophagy)
Electron transport generates reactive oxygen species,
which can damage DNA and oxidize proteins
How does a cell deal with damaged or aged mitochondria?
1. Repair: Fission and fusion
2. Send vesicles with damaged components to
peroxisomes and lysosomes
3. Destroy mitochondria through autophagy (mitophagy)
Mitochondria can fuse together and divide
by fission
Genes controlling mitochondrial fission and fusion are required for mitochondrial
morphology and function
Emerging functions of mammalian mitochondrial fusion and fission. Chen H, Chan DC. Hum Mol
Genet. 2005 Oct 15;14
Genes controlling mitochondrial fission and fusion are required for mitochondrial
morphology and function
Emerging functions of mammalian mitochondrial fusion and fission. Chen H, Chan DC. Hum Mol
Genet. 2005 Oct 15;14
Genes controlling mitochondrial fission and fusion are required for mitochondrial
morphology and function
respiratory defects in
fusion mutants
Emerging functions of mammalian mitochondrial fusion and fission. Chen H, Chan DC. Hum Mol
Genet. 2005 Oct 15;14
Disruption of fusion results in mitochondrial heterogeneity and dysfunction. Chen H, Chomyn A,
Chan DC. J Biol Chem. 2005 Jul 15;280(28):26185-92.
Knockout of the mitochondrial fusion genes Mfn1 and Mfn2 in the cerebellum of
mice results in...
1. abnormal mitochondria
with defective OxPhos
Mitochondrial fusion protects against neurodegeneration in the cerebellum. Chen H, McCaffery
JM, Chan DC. Cell. 2007 Aug 10;130(3):548-62.
Knockout of the mitochondrial fusion genes Mfn1 and Mfn2 in the cerebellum of
mice results in...
1. abnormal mitochondria
with defective OxPhos
2. Death of Purkinje cells and cerebellar degeneration
Mitochondrial fusion protects against neurodegeneration in the cerebellum. Chen H, McCaffery
JM, Chan DC. Cell. 2007 Aug 10;130(3):548-62.
Knockout of the mitochondrial fusion genes Mfn1 and Mfn2 in the cerebellum of
mice results in...
1. abnormal mitochondria
with defective OxPhos
2. Death of Purkinje cells and cerebellar degeneration
3.Impaired cerebellar function
Mitochondrial fusion protects against neurodegeneration in the cerebellum. Chen H, McCaffery
JM, Chan DC. Cell. 2007 Aug 10;130(3):548-62.
How does a cell deal with damaged or aged mitochondria?
1. Repair: Fission and fusion
2. Send vesicles with damaged components to
peroxisomes and lysosomes
3. Destroy mitochondria through autophagy (mitophagy)
Adding glucose oxidase, which generates ROS, to HeLa or Cos7 cells
generates vesicles containing mitochondrial outer membrane proteins
Mitochondriaderived
vesicles (MDVs)
fragmentation
A vesicular transport pathway shuttles cargo from mitochondria to lysosomes. Soubannier V,
McLelland GL, Zunino R, Braschi E, Rippstein P, Fon EA, McBride HM. Curr Biol. 2012 Jan
24;22(2):135-41
Some MDVs colocalize with lysosome markers
Tom20 = mitochondrial outer membrane; Oct = mitochondrial inner membrane; Lamp1 = lysosomal
A vesicular transport pathway shuttles cargo from mitochondria to lysosomes. Soubannier V,
McLelland GL, Zunino R, Braschi E, Rippstein P, Fon EA, McBride HM. Curr Biol. 2012 Jan
24;22(2):135-41
Some MDVs colocalize with lysosome markers
Tom20 = mitochondrial outer membrane; Oct = mitochondrial inner membrane; Lamp1 = lysosomal
Blocking lyososomal degradation (bafilomycin/PA) increases MDVs
A vesicular transport pathway shuttles cargo from mitochondria to lysosomes. Soubannier V,
McLelland GL, Zunino R, Braschi E, Rippstein P, Fon EA, McBride HM. Curr Biol. 2012 Jan
24;22(2):135-41
How does a cell deal with damaged or aged mitochondria?
1. Repair: Fission and fusion
2. Send vesicles with damaged components to
peroxisomes and lysosomes
3. Destroy mitochondria through autophagy
(mitophagy)
Pink1 and Parkin: Two proteins associated with Parkinson’s disease
What do PINK1 and Parkin do?
PINK1 is a kinase and Parkin is an E3 Ubiquitin ligase
1. With what cellular process are PINK1 and Parkin
associated?
2. Where in the cell do PINK1 and Parkin localize?
3. What proteins are phosphorylated by PINK1?
4. What proteins are tagged for degradation by Parkin?
In Drosophila, PINK1 is localized to mitochondria
Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Clark IE,
Dodson MW, Jiang C, Cao JH, Huh JR, Seol JH, Yoo SJ, Hay BA, Guo M. Nature. 2006 Jun
29;441(7097):1162-6.
In Drosophila, PINK1 is localized to mitochondria
And Mitochondria are abnormal in PINK1 mutants
Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Clark IE,
Dodson MW, Jiang C, Cao JH, Huh JR, Seol JH, Yoo SJ, Hay BA, Guo M. Nature. 2006 Jun
29;441(7097):1162-6.
Parkin mutants have a similar phenotype as PINK1 mutants and Parkin
overexpression partially suppresses PINK1 mitochondrial defects
PINK1 mutants overexpressing Parkin
PINK1
Parkin
Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Clark IE,
Dodson MW, Jiang C, Cao JH, Huh JR, Seol JH, Yoo SJ, Hay BA, Guo M. Nature. 2006 Jun
29;441(7097):1162-6.
What do PINK1 and Parkin do?
PINK1 is a kinase and Parkin is an E3 Ubiquitin ligase
1. With what cellular process are PINK1 and Parkin
associated?
2. Where in the cell do PINK1 and Parkin localize?
3. What proteins are phosphorylated by PINK1?
4. What proteins are tagged for degradation by Parkin?
Mitochondrial damage recruits Parkin to mitochondria
Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live
Cortical Neurons (2012). Cai Q, Zakaria HM, Simone A, Sheng ZH. Curr Biol. 22(6):545-52.
Mitochondria associated with Parkin accumulate near the cell body;
likely because of a defect in anterograde movement on microtubules
Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live
Cortical Neurons (2012). Cai Q, Zakaria HM, Simone A, Sheng ZH. Curr Biol. 22(6):545-52.
Damage causes mitochondria to accumulate in autophagosomes and
lysosomes
Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live
Cortical Neurons (2012). Cai Q, Zakaria HM, Simone A, Sheng ZH. Curr Biol. 22(6):545-52.
Parkin-associated mitochondria are eliminated by autophagy
Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live
Cortical Neurons (2012). Cai Q, Zakaria HM, Simone A, Sheng ZH. Curr Biol. 22(6):545-52.
Parkin-associated mitochondria are eliminated by autophagy
Green= Parkin-YFP, Red=dsRED-mito
CCCP added
Spatial Parkin Translocation and Degradation of Damaged Mitochondria via Mitophagy in Live
Cortical Neurons (2012). Cai Q, Zakaria HM, Simone A, Sheng ZH. Curr Biol. 22(6):545-52.
Is Pink1 recruitment sufficient to promote degradation?
An inducible protein association system: In the presence of the drug rapalog protein
gets recruited to mitochondria
Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment
and Activation of the E3 Ligase Parkin. Lazarou M, Jin SM, Kane LA, Youle RJ. Dev Cell. 2012 Feb
14;22(2):320-33
Is PINK1 recruitment sufficient to promote degradation?
This system can be used to recruit PINK1 to peroxisomes and lysosomes
Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment
and Activation of the E3 Ligase Parkin. Lazarou M, Jin SM, Kane LA, Youle RJ. Dev Cell. 2012 Feb
14;22(2):320-33
Is PINK1 recruitment sufficient to promote degradation?
Artificial recruitment of PINK1 promotes mitophagy and pexophagy
Role of PINK1 Binding to the TOM Complex and Alternate Intracellular Membranes in Recruitment
and Activation of the E3 Ligase Parkin. Lazarou M, Jin SM, Kane LA, Youle RJ. Dev Cell. 2012 Feb
14;22(2):320-33
Model:
1. Damaged mitochondria recruit PINK1 to their surface, which, in turn, recruits
Parkin.
2. PINK1 phosphorylates Miro and Parkin degrades it, halting anterograde
mitochondrial transport
3. PINK1/Parkin recruit mitophagy machinery, leading to the elimination of
damaged mitochondria
PINK1 and Parkin flag Miro to direct mitochondrial traffic. Kane LA, Youle RJ. Cell. 2011 Nov
11;147(4):721-3.
Model:
1. Damaged mitochondria recruit PINK1 to their surface, which, in turn, recruits
Parkin.
2. PINK1 phosphorylates Miro and Parkin degrades it, halting anterograde
mitochondrial transport
3. PINK1/Parkin recruit mitophagy machinery, leading to the elimination of
damaged mitochondria
Hypothesis:
The accumulation of damaged mitochondria
causes neuronal death and Parkinson’s disease
PINK1 and Parkin flag Miro to direct mitochondrial traffic. Kane LA, Youle RJ. Cell. 2011 Nov
11;147(4):721-3.
Lysosomes, peroxisomes, mitochondria
1. Lysosomes are low pH membranous organelles containing
hyrdrolases that digest phagocytosed and autophagosed material
2. Proteins destined for the lysosome are tagged by mannose-6phosphate in the Golgi and sorted in endosomes
3. Peroxisomes are organelles that execute a variety of metabolic
reactions
4. Peroxisomal proteins are synthesized in the cytoplasm
5. In the presence of oxygen mitochondria convert pyruvate into Acetyl
CoA, generate NADH through the TCA cycle, and create ATP via
electron transport and the function of ATP synthase
6.Knocking out genes required for mitochondrial fission and fusion
leads to accumulation of damaged mitochondria and cell death
7. Damaged material can bud from mitochondria and be transported
to lysosomes and peroxisomes
8. PINK1 and Parkin mutations are responsible for Parkinson’s
disease and regulate autophagy of mitochondria.