Plant Cell, Development & Ultrastructure - Botanik in...

PCDU – Lecture

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Plant Cell, Development & Ultrastructure

Plant Cell Biology Labs

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Endomembranes - Dictyosome / Golgi Apparatus

Golgi Stack = Dictyosom


Check the „ultimate golgi video/song“ http:/www.brookes.ac.uk/plant_endomembrane

Golgi Apparatus

Dictyosome (Reticulate Body)

Trans-Side

Cis-Side


Golgi Apparatus

Intercisternal Filaments


100nm

Hawes 2005

Tethering Factors

Barringa et al. 2009

Golgin

Giantin

Golgi Apparatus


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


Heterotrimeric G-Proteins

Retrieval of

ER-resident Proteins

Karp 1999

KDEL

Lys – Lysin

Asp – Asparagin

Glu – Glutaminsäure

Leu – Leucin

HDEL

(Plants)


Endoplasmic Reticulum

Retrieval of

ER-resident Proteins

Karp 1999

KDEL

Lys – Lysin

Asp – Asparagin

Glu – Glutaminsäure

Leu – Leucin

HDEL

bei Pflanzen


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


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


Brefeldin A

Effects of BFA on the Endomembrane System

Nebenführ (2002) Plant Physiol. 130(3):1102-1108.


Brefeldin A

BFA inhibits ER Export

BFA-Induced Compartments izmb.uni-bonn.de izmb.uni-bonn.de

PCR BFA Compartments

Endocytosis

Samaj et al., 2004

BFA inhibits ER Export

500nm Grebe et al., 2003

25µM BFA, 30min


BFA-Induced Compartments

12

Clathrin-Coated Vesicles for Vesicle Recycling

36 Triskelions = 12 Pentagons + 6 Hexagons


Clathrin

Adaptor Proteins Confer Membrane Domain Specificity

Receptor-

Coupled

Endocytosis

192 kDa

25-30 kDa


AP-1 AP-2

homology

AP-1

AP-2

Clathrin

Anti-Transferrin

15nm gold conjugate


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

Molecular Structure of Clathrin


Clathrin

100kDa Mechanochemical Enzyme:

Involved in Formation of Coated Vesicles

Synaptotagmin

Clathrin

Amphiphysin Dynamin


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


Dynamin

Intracellular Motility of Golgi Stacks in Animals Cells

is Dependent on the Microtubule Cytoskeleton


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


Dictyosomes Dictyosomes

plus actin

ER

© Chris Hawes

Golgi Stacks

(Dictyosomes)

Golgi Stacks in Plant Cells

Secretory Pathway to Vacuoles


Prevacuolar Compartment

PVC


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


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



Synthetic Functions of Golgi Stacks

Hemicelluloses Pectins

Aktivierte Monosaccharide

Transporter


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


The SNARE Hypothesis

Explains Targeted Vesicle Fusion


SNARE Hypothesis

Exocyst

(v-snare)

(v-snare)

(t-snare) tethering

complex

Kiss & Run


Vesicle Fusion with Plasma Membrane


Bai & Chapman 2004

SYT1 Controls Membrane Repair in Plant Cells

Synaptotagmin

Synaptotagmins in Plants

Microbodies/ Peroxisomes


1

Source for H2O2 in Catabolic pathways


Katalase

Uricosomen in Stickstoff-speichernden

Geweben

Glyoxysomen in Stickstoff-speichernden

Geweben

Peroxisomen in Blattgewebe

2

Nitrogen storage in uricosomes


3

Histochemical test for catalase


DAB

H2O2

+ DAB

Diaminobenzidin

4


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



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

6


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



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

8

Protein import into peroxisomes


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.

9





PEX7

PTS1 is recognized by

seven TPR repeats at the

contact domain of PEX5

PTS1: C-terminal tripeptide „SKL“


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.

10






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





PTS = peroxisomal

targeting signal

PEX5-PEX7

glyoxysomal receptor

PEX6-PEX7


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

12





PTS = peroxisomal

targeting signal

PEX5-PEX7


PEX6-PEX7


13



Evolutionary comparison of the

PEX transport machinery

BROWN, L-A & BAKER, A (2008) Molec.Membr.Biol. 25(5): 363-375.

PTS1 PTS2

PEX5/PEX7

14

Peroxisomes


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

15

Peroxisomes


DLP1 = dynamin-like protein 1

responsible for peroxisomal fission

Proliferation of Peroxisomes/Glyoxysomes

pre-peroxisome

1

2

3

4

16


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

Plant Cell, Development & Ultrastructure - Botanik in...

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