1. 6. Ca 2+ -ATPases, another group of P-type ATPase, are distributed among various plant membranes. PM Vacuole Ca 2+ ATP ADP+Pi ER Ca 2+ ATP ADP+Pi Golgi Ca 2+ ATP ADP+Pi Ca 2+ ATP ADP+Pi Nucleus H+H+ Ca 2+ Ca 2+ /H+ antiporter Ca 2+ Ca 2+ channel Ca 2+ Ca 2+ channel Ca 2+ Ca 2+ channel Ca 2+ Ca 2+ channel

2. Autoinhibited Calcium ATPases (ACAs) NH 2 COOH CaM-Binding Auto- inhibitor Asp-P ATP-binding Ca 2+ 14510 NH 2 COOH Asp-P ATP-binding Ca 2+ 14510 ER-type Calcium ATPases (ECAs) Structural Characteristic of Ca 2+ -ATPases

3. ACA9 ACA13 ACA1 ACA2 ACA7 ACA4 ACA11 ACA10 ACA8 ACA12 ER group Vacuole group PM group Unknown group Autoinhibited Ca 2+ - ATPase (ACA)

4. Free GFP (Cytosol) 35S-ACA8-GFP (PM) 35S-ACA11-GFP (Vacuole) GFPRFPDICMerge Localization of ACA11 in Arabidopsis Protoplast

5. Phenotype of vacuole Ca 2+ pump mutants (aca4/aca11)

6. Complementation of the aca11/aca4 mutant with the ACA11 gene

7. WT KO Bright UV Lactophenol cleaning (Phenolic compound) Anilline blue staining (callose) Bright UV Cell death in aca4/aca11 is a HR-like PCD

8. W.T x60 #316 V V V VVV EM Photos of aca4/aca11 mutants

9. Recovery phenotype of aca11/aca4 mutant by phosphate Transfer to soil after growth in MSO media for 10 days

10. chloroplast ER Nucleus Golgi Ca 2+ PiCa 2+ PiCa 2+ PiCa 2+ PiCa 2+ PiCa 2+ PiCa 2+ Pi Cell Death Signal??? VPE activation membrane collapse Cell Death Plasma membrane Ca 2+ chloroplast PiCa 2+ PiCa 2+ PiCa 2+ PiCa 2+ chloroplast Hypothesis

11. 7. Vacuolar and other membranes are energized through vacuolar H+-ATPase Structural model of V-type H+-ATPase V 1 sector V 0 sector pH 7.5 pH 5.5 (pH 3.0)

12.  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

13. The H+-ATPase are potently and specifically inhibited by the macrolide antibiotic bafilomycin A1

14. 8. The plant vacuolar membrane also possesses a unique H+-pumping inorganic pyrophosphatase (H+-ppase) PPi  2 pi

15. 9. ABC-type pumps are emerging as major players in sequestration of amphipathic metabolites and xenotoxics into the vacuole ABC: ATP binding cassette

16. Transport of a glutathione-conjugated xenotoxic and a chlorophyll Catabolite by AtMRP2, and ABC transporter from Arabidpsis.

17. Cd 2+ detoxification pathway in yeast and plant Glu+Cys GCS γ-GluCys +Gly GS GSH PCS Cd 2+ PC LMW HMT1 Cd 2+ Cd(GSH) 2 YCF1 Cd- PC LMW Cd- PC sulfide cytoplasm vacuole HMW Cd- PC Christopher S.C. et al (2000)

18. 3.4. Carriers

19. 1.Carriers exhibit Michaelis-Menten kinetics that indicate conformational changes during transport. Carriers exhibit saturation kinetics.

20. Carriers undergo conformational changes during transport (ex, The activity of carrier C)

21. 2. Carriers translocate a wide variety of inorganic and small organic solutes with high specificity. 1) Inorganic nutrients: NH 4+, NO 3-, P i, K +, SO 4 2-, Cl - 2) Organic solutes: sugars, amino acids, purine and pyrimidine bases

22. 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)

23. 3. Most plant carriers are energized by coupling to pmf.

24. 4. Molecular identification of carriers defines them as members of the major facilitator superfamily Functional analysis 1.Yeast complementation 2.Protein expression in Oocytes

25. Observation of plant carriers expressed in heterologous systems can provide into carrier function

26. Structural model illustrating the orientation of a generalized carrier in a membrane

27. 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

28. 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

29. 6. Regulation of carrier activity 1) By transcriptional control 2) By post-translational control

30. Low K + : 40 uM, High K + : 2 mM Different transcription of AtKUP2 and AtKUP3

31. 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

32. Acetabularia, a marine algae