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