Post on 17-Sep-2018
PCDU – Lecture
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Plant Cell, Development & Ultrastructure
Plant Cell Biology Labs
Download at:
http://goo.gl/111Tha
Endomembranes - Dictyosome / Golgi Apparatus
Golgi Stack = Dictyosom
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Check the „ultimate golgi video/song“ http:/www.brookes.ac.uk/plant_endomembrane
Golgi Apparatus
Intercisternal Filaments
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100nm
Hawes 2005
Tethering Factors
Barringa et al. 2009
Golgin
Giantin
Golgi Apparatus
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Tethering Factors
GRIB-domain = C-terminal Arf-binding domain
named after the first 4 identified members
GRAB-domain = GRIP-related Arf-binding domain
GA 1 = GRAB-associated
Plant Golgin - Candidate Proteins Model of Golgin/G-Protein Interaction
Arl = Arf-like
Arf = ADP-ribosylation factor
Rab = Ras-related in brain
Ras =rat sarcoma onkogen
N-Terminus C-Terminus
Golgi Apparatus
GTP Cycle in Small G-Proteins
GEF = Guanidin Nucleotide Exchange Factor
GAP = GTPase Activating Protein
GDI = Guanine Nucleotide Dissociation Inhibitor
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Heterotrimeric G-Proteins
Retrieval of
ER-resident Proteins
Karp 1999
KDEL
Lys – Lysin
Asp – Asparagin
Glu – Glutaminsäure
Leu – Leucin
HDEL
(Plants)
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Endoplasmic Reticulum
Retrieval of
ER-resident Proteins
Karp 1999
KDEL
Lys – Lysin
Asp – Asparagin
Glu – Glutaminsäure
Leu – Leucin
HDEL
bei Pflanzen
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Regulated by ARF/GEF
G-Protein
Endoplasmic Reticulum
COPII-Coated Vesicles
Nickel (1999) Protoplasma 207: 115-124
ARF/GEF
GEF = Guanidin Nucleotide Exchange Factor
BFA
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Blickford et al. 2004
ARF GEFs are Targets of Brefeldin A
Fungal Inhibitor Brefeldin A (BFA)
BFA is a fatty acid metabolite (makrocyclic lactone) from Penicillum brefeldianum. In the
fungus it is active as an antiviral agent. In animal and plant cells it inhibits anterograde
vesicle transport from ER to Golgi. Golgi stacks disappear with the consequence that
exocytosis is abolished. Molecular target is a GTP/GDP-Exchange Factor (GEF) that
activates small G-proteins involved in the formation of COP-II coated vesicles.
C16H24O4
MW: 280.36 Da
1, 13-dihydroxy-6-methyl-4H-cyclopentoxacyclotridecin-4-on
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Brefeldin A
Effects of BFA on the Endomembrane System
Nebenführ (2002) Plant Physiol. 130(3):1102-1108.
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Brefeldin A
PCR BFA Compartments
Endocytosis
Samaj et al., 2004
BFA inhibits ER Export
500nm Grebe et al., 2003
25µM BFA, 30min
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BFA-Induced Compartments
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Clathrin-Coated Vesicles for Vesicle Recycling
36 Triskelions = 12 Pentagons + 6 Hexagons
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Clathrin
Adaptor Proteins Confer Membrane Domain Specificity
Receptor-
Coupled
Endocytosis
192 kDa
25-30 kDa
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AP-1 AP-2
homology
AP-1
AP-2
Clathrin
Anti-Transferrin
15nm gold conjugate
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Keyel et al. 2008
Transferrin is an Fe-transporting
Glycoprotein that is recognized
by the transferrin-receptor and
taken up into the cell by receptor-
coupled endocytosis
100kDa Mechanochemical Enzyme:
Involved in Formation of Coated Vesicles
Synaptotagmin
Clathrin
Amphiphysin Dynamin
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Adaptin Complex
Karp 1999
Dynamin
Bottomley (1999) Curr. Biol. 9: R301-R304
Recruitment of Dynamin to Membranes
Pleckstrin-Domain
PH = Plekstrin-Homology Domain
GED = GTPase regulatory Domain
PRD = Prolin rich Domain
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Dynamin
Intracellular Motility of Golgi Stacks in Animals Cells
is Dependent on the Microtubule Cytoskeleton
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Dictyosomes Dictyosomes
plus actin
Microtubules in
mouse fibroblasts, © Jan Schmoranzer
http://www.maths.bris.ac.uk
Golgi in HeLa cell
© Abcam plc 2008
Golgi Stacks in Animal Cells
Intracellular Motility of Golgi Stacks (Dictyosomes) in
Plant Cells is Dependent on the Actin Cytoskeleton
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Dictyosomes Dictyosomes
plus actin
ER
© Chris Hawes
Golgi Stacks
(Dictyosomes)
Golgi Stacks in Plant Cells
Prevacuolar Compartment
PVC
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ESCRT = Endosomal Sorting Complex Required for Transport
MVB = Multi-Vesicular Body
Retromer = Rretrograde Transport Complex to the TGN
Otegui & Spitzer 2008
Prevacuolar Compartment – PVC
Multivesicular Body – MVB
ESCRT Complex - ESCRT
Retromer Complex izmb.uni-bonn.de izmb.uni-bonn.de
Retromer = Pentameric Protein Complex for the Retrieval of VSR
VSR = Vacuolar Sorting Receptor
26/29/35 = VPS proteins (Vacuolar Protein Sorting Proteins)
SNX = Sorting Nexin
PI3P = Phosphatidylinositol 3-Phosphate
coiled-coil
PI-3-P-binding
Transport of Cargo from MVB Back to TGN
Schellmann & Pimpl 2009
Dynamics within Golgi Stack:
Stationary Cisternae Model
versus Vesicle Shuttle Model
Buchanan et al. 2000
Intercisternal
elements
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Dynamics within Golgi Stack
Dictyosomes in Coccolith Algae
Support the Cisternal Progression Model
Cross-sectional model of coccolith formation
in the Golgi of P. carterae.
(1) Polysaccharide scale formation
(2) Mineral deposition
(3) Mature Coccosphere
P = Ca2+-containing particles
Coccolithophora
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Dynamics within Golgi Stack
Synthetic Functions of Golgi Stacks
Hemicelluloses Pectins
Aktivierte Monosaccharide
Transporter
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ATP
ADP
Uncoating ATPases
NSF= Nethylmaleimid-Sensitive Factor (triple helix resolving ATPase)
SNAP= soluble N-ethylmaleimide-sensitive-factor Attachment Protein
SNARE= soluble N-ethylmaleimide-sensitive-factor Attachment-Protein REceptor
NSF
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The SNARE Hypothesis
Explains Targeted Vesicle Fusion
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SNARE Hypothesis
Exocyst
(v-snare)
(v-snare)
(t-snare) tethering
complex
Kiss & Run
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Vesicle Fusion with Plasma Membrane
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Bai & Chapman 2004
SYT1 Controls Membrane Repair in Plant Cells
Synaptotagmin
Synaptotagmins in Plants
Source for H2O2 in Catabolic pathways
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Katalase
Uricosomen in Stickstoff-speichernden
Geweben
Glyoxysomen in Stickstoff-speichernden
Geweben
Peroxisomen in Blattgewebe
2
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Import of peroxisomal membrane proteins
Peroxisomal membrane proteins (PMPs) enter the ER, are sequestered in specialized regions and bud from the ER to form peroxisomal pre-
compartments. These become capable of importing PTS1/PTS2 containing proteins and develop into mature peroxisomes or fuse with existing
peroxisomes. Enlarged peroxisomes can divide by fission.
Lipid import
mature peroxisome pre-peroxisome
5
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Import of peroxisomal membrane proteins
NBF = Nucleotide binding fold
TMD = Transmembrane domain
ABC = ATP-binding Cassette
peroxisomal ABC-transporter
CTS = comatose
(mutant seeds do not germinate)
Fatty acid import by peroxisomal ABC-transporters
Dietrich et al. 2009
cytoplasm
lumen
yeast &
mammals plants
NBF
6 TMDs
ATP
There are ca. 130 genes in the Arabidopsis Genome coding for ABC-proteins
dimer fused dimer
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Import of metabolites into peroxisomes
Fulda, M. et al. (2004) Plant Cell 16: 394–405.
Fatty acid import by ABC-transporters
Acyl-CoA-handover
PXA1/2
7
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Import of peroxisomal membrane proteins
Peroxisomal membrane proteins (PMPs) enter the ER, are sequestered in specialized regions and bud from the ER to form peroxisomal
precompartments. These become capable of importing PTS1/PTS2 containing proteins and develop into mature peroxisomes or fuse with existing
peroxisomes. Enlarged peroxisomes can divide by fission.
PTS1/PTS2 = Peroxisome-targeting signals
PTS1: C-terminal tripeptide „SKL“
PTS2: N-terminal R(X6)(H/Q)ALF
Lipid import
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Protein import into peroxisomes
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Luminal (Matrix) Proteins:
Role of cytosolic receptors pex5/6 and pex7
PEX7
PTS1: C-terminal tripeptide „SKL“ PTS2: N-terminal R(X6)(H/Q)ALF
Stanley et al. (2007)
FEBS Lett 581: 4795–4802.
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Protein import into peroxisomes
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Luminal (Matrix) Proteins:
Role of cytosolic receptors pex5/6 and pex7
PEX7
PTS1 is recognized by
seven TPR repeats at the
contact domain of PEX5
PTS1: C-terminal tripeptide „SKL“
Stanley et al. (2007)
FEBS Lett 581: 4795–4802.
The tetratrico peptide repeat
(TPR) is a structural motif of 3-16
tandem-repeats of 34 amino acids
residues each. It mediates protein-
protein interactions and the
assembly of multi-protein
complexes.
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Protein import into peroxisomes
izmb.uni-bonn.de izmb.uni-bonn.de
Luminal (Matrix) Proteins:
Role of cytosolic receptors pex5/6 and pex7
Stanley et al. (2007)
FEBS Lett 581: 4795–4802.
The tetratrico peptide repeat
(TPR) is a structural motif of 3-16
tandem-repeats of 34 amino acids
residues each. It mediates protein-
protein interactions and the
assembly of multi-protein
complexes.
11
Protein import into peroxisomes
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Luminal (Matrix) Proteins:
Role of cytosolic receptors pex5/6 and pex7
PTS = peroxisomal
targeting signal
PEX5-PEX7
glyoxysomal receptor
PEX6-PEX7
glyoxysomal receptor
PEX7
PTS2 is recognized by a
beta-propeller domain
(Seven WD40 repeats) at the
contact site of PEX7
PTS2: N-terminal
alpha-helical motif
R(X6)(H/Q)ALF
W – Tryptophan
D – Aspartic acid
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Protein import into peroxisomes
izmb.uni-bonn.de izmb.uni-bonn.de
Luminal (Matrix) Proteins:
Role of cytosolic receptors pex5/6 and pex7
PTS = peroxisomal
targeting signal
PEX5-PEX7
glyoxysomal receptor
PEX6-PEX7
glyoxysomal receptor
13
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Protein import into peroxisomes
Evolutionary comparison of the
PEX transport machinery
BROWN, L-A & BAKER, A (2008) Molec.Membr.Biol. 25(5): 363-375.
PTS1 PTS2
PEX5/PEX7
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Peroxisomes
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docking complex RING finger complex
PEX4 = ubiquitin conjugating enzyme
PEX2/12/10 = ubiquitin ligase
PEX22=anchoring protein for PEX4
PEX1/6 = ATPase peroxins
removal of peroxisomal
targeting sequences
Targeting & recycling of PEX5 & PEX7 via mono-ubiquitination
mono-ubiquitination
poly-ubiquitination
followed by protein
degradation on 26S
proteasomes
de-ubiquitination
releasing complex
*
* RING finger domain
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Peroxisomes
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DLP1 = dynamin-like protein 1
responsible for peroxisomal fission
Proliferation of Peroxisomes/Glyoxysomes
pre-peroxisome
1
2
3
4
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Peroxisomes
Peroxisomal metabolism results in the generation of signalling
molecules, which fall into four broad catergories.
Nyathi & Baker (2006) Biochimica et Biophysica Acta 1763: 1478–1495
ASC: ascorbate
DHA: dehydroascorbate
GSH: reduced glutathione
GSSG: oxidized glutathione
GSNO: S-nitroso-glutathione 17