6. Ca 2+ -ATPases, another group of P-type ATPase, are distributed among various plant membranes. PM...
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Transcript of 6. Ca 2+ -ATPases, another group of P-type ATPase, are distributed among various plant membranes. PM...
6. Ca2+-ATPases, another group of P-type ATPase, are distributed among various plant membranes.
PM
Vacuole
Ca2+
ATP
ADP+Pi
ERCa2+
ATP
ADP+Pi
Golgi
Ca2+
ATP
ADP+Pi
Ca2+
ATP
ADP+Pi
Nucleus
H+
Ca2+
Ca2+ /H+ antiporterCa2+
Ca2+ channel
Ca2+
Ca2+ channel
Ca2+
Ca2+ channel
Ca2+Ca2+ channel
Autoinhibited Calcium ATPases (ACAs)
NH2
COOH
CaM-Binding
Auto-inhibitor
Asp-P
ATP-binding
Ca2+
1 4 5 10
NH2
COOH
Asp-P
ATP-binding
Ca2+
1 4 5 10
ER-type Calcium ATPases(ECAs)
Structural Characteristic of Ca2+-ATPases
ACA9
ACA13
ACA1
ACA2ACA7
ACA4
ACA11
ACA10
ACA8
ACA12
ER group
Vacuole group
PM group
Unknown group
Autoinhibited Ca2+- ATPase (ACA)
Free GFP(Cytosol)
35S-ACA8-GFP(PM)
35S-ACA11-GFP(Vacuole)
GFP RFP DIC Merge
Localization of ACA11 in Arabidopsis Protoplast
Phenotype of vacuole Ca2+ pump mutants (aca4/aca11)
Complementation of the aca11/aca4 mutant with the ACA11 gene
WT
KO
Bright UV
Lactophenol cleaning(Phenolic compound)
Anilline blue staining(callose)
Bright UV
Cell death in aca4/aca11 is a HR-like PCD
W.T W.T x60
#316 #316 #316
V
V
V
VVV
EM Photos of aca4/aca11 mutants
Recovery phenotype of aca11/aca4 mutant by phosphate
Transfer to soil after growth in MSO media for 10 days
chloroplast
ER
Nucleus
Golgi
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
PiCa2+
Ca2+Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
PiCa2+
PiCa2+
PiCa2+
PiCa2+PiCa2+
Pi
Pi
Cell Death Signal???
VPE activation
membrane collapse
Cell DeathPlasma membrane
Ca2+
Ca2+
Ca2+ Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+
Ca2+Ca2+
Ca2+
Ca2+
chloroplast
PiCa2+
PiCa2+
PiCa2+
PiCa2+
chloroplast
Hypothesis
7. Vacuolar and other membranes are energized through vacuolar H+-ATPase
Structural model of V-type H+-ATPase
V1 sector
V0 sector
pH 7.5
pH 5.5(pH 3.0)
Plant vacuole contains a highly acidic solution, with pH 5.5.
Proton pumping into the vacuolar lumen not only energizes the membrane for carrier-mediated transport but also generates the low pH of the vacuole.
An acidic lumemal pH is thought to contribute to vesicle trafficking And protein targeting.
These enzymes are present in membranes from th ER, Golgi, and coated vesicles of plant cells
Function of V-type H+-ATPases
The H+-ATPase are potently and specifically inhibited by the macrolide antibiotic bafilomycin A1
8. The plant vacuolar membrane also possesses a unique H+-pumping inorganic pyrophosphatase (H+-ppase)
PPi 2 pi
9. ABC-type pumps are emerging as major players in sequestration of amphipathic metabolites and xenotoxics into the vacuole
ABC: ATP binding cassette
Transport of a glutathione-conjugated xenotoxic and a chlorophyll Catabolite by AtMRP2, and ABC transporter from Arabidpsis.
Cd2+ detoxification pathway in yeast and plant
Glu+Cys
GCS
γ-GluCys +Gly
GS
GSH
PCS
Cd2+
PC
LMW
HMT1
Cd2+
Cd(GSH)2
YCF1
Cd- PCLMW
Cd- PC
sulfide
sulfide
cytoplasm
vacuole
HMWCd- PC
Christopher S.C. et al (2000)
3.4. Carriers
1. Carriers exhibit Michaelis-Menten kinetics that indicate conformational changes during transport.
Carriers exhibit saturation kinetics.
Carriers undergo conformational changes during transport (ex, The activity of carrier C)
2. Carriers translocate a wide variety of inorganic and small organic solutes with high specificity.
1) Inorganic nutrients: NH4+, NO3-, Pi, K+, SO42-, Cl-
2) Organic solutes: sugars, amino acids, purine and pyrimidine bases
Functions of carriers
1. At plasma membrane, - nutrient uptake - the mobilization and storage of metabolites.
2. At endomembrane, - sequestration of ions (Na+, Ca2+, Mg2+, No3-, sugars, aa)
3. Most plant carriers are energized by coupling to pmf.
4. Molecular identification of carriers defines them as members of the major facilitator superfamily
Functional analysis
1. Yeast complementation
2. Protein expression in Oocytes
Observation of plant carriers expressed in heterologous systems can provide into carrier function
Structural model illustrating the orientation of a generalized carrier in a membrane
Localization of the sucrose transporter SUC2 to companion cells of the phloem
A. Immunofluorescent localization of SUC2 in Arabidopsis stems.
B. Same section as in A but viewed with transmitted light.
P: phloem X: xylem
Localization of the sucrose transporter SUT1 to sieve elements
A. Immunofluorescent localization of SUT1 in a longitudinal section of a potato stems.
B. Silver-enhanced Immunogold localization of SUT1 in cross-section of a potato petiole
sp: sieve plate n: nucleus cc: companion cells
6. Regulation of carrier activity
1) By transcriptional control
2) By post-translational control
Low K+: 40 uM,
High K+: 2 mM
Different transcription of AtKUP2 and AtKUP3
7. In some cases, ion-coupled solute transport involves
Na+ rather than H+
1. Na+ symport have been found.
2. Na+coupling of K+ transport has been proved by a wheat cDNA
a. Uptake of NO3- and some amino acids is Na+-dependent
b. uptake of K+ at micromolar concentration is also Na+-dependent
Acetabularia, a marine algae