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First published online March 14, 2008
Journal of Experimental Biology 211, 1102-1108 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.013672
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Biochemical and functional characterization of the actin-binding activity of the B subunit of yeast vacuolar H+-ATPase

Jian Zuo1, Sandra Vergara1, Shinya Kohno2 and L. Shannon Holliday1,3,*

1 Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610, USA
2 Department of Orthodontics and Craniofacial Developmental Biology, Hiroshima University, Hiroshima, Japan
3 Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610, USA


Figure 1
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  		Fig. 1. Yeast B subunit binds F-actin. F-actin was diluted to 800 nmol l–1 and stabilized with 5 µmol l–1 phalloidin, mixed with various concentrations of Vma2p-MBP, and subjected to ultracentrifugation. Pellets and supernatants were collected, separated by SDS-PAGE and stained with Coomassie Brilliant Blue (A). M, markers (top to bottom; 110 kDa, 67 kDa, 43 kDa); lane 1, no actin, 2 µmol l–1 Vma2p-MBP; lane 2, 0.8 µmol l–1 F-actin, 0.4 µmol l–1 Vma2p-MBP; lane 3, 0.8 µmol l–1 F-actin, 0.8 µmol l–1 Vma2p-MBP; lane 4, 0.8 µmol l–1 F-actin, 1.2 µmol l–1 Vma2p-MBP; lane 5, 0.8 µmol l–1 F-actin, 1.6 µmol l–1 Vma2p-MBP; lane 6, 0.8 µmol l–1 F-actin, 2.0 µmol l–1 Vma2p-MBP. The pellets were loaded with twice the relative amount of protein compared with supernatants. (B) Actin was polymerized and diluted into solutions containing phalloidin (5 µmol l–1) and yeast B-MBP as described under Materials and methods. The final actin concentration, indicated by the broken line, was 800 nmol l–1. Following incubation and centrifugation, the supernatants and pellets were collected and the amount of fusion protein present was determined by densitometry of Coomassie-stained gels. (C) Varying amounts of Vma2p-MBP were added to F-actin and subjected to high speed centrifugation as described under Materials and methods. Pellets and supernatants were analyzed by SDS-PAGE and densitometry was used to determine the amount of protein in the Coomassie-stained gels. The Kd was calculated based on the finding that the fusion protein binding saturated at 1 mol of fusion protein per mol of actin filament subunit (established in A and B).

 

Figure 2
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  		Fig. 2. Mutations in the profilin-like motif of yeast B subunit reduce or eliminate actin-binding activity. (A) Alterations in the profilin-like motif made and analyzed in the current study. Amino acids 35–45 in wild-type Vma2p, Vma2pPhe and Vma2pArch are compared. (B) Haine's plot of Vma2pPhe-MBP binding to F-actin. (C) Vma2pArch-MBP does not bind F-actin: Lane 1, supernatant and lane 2 pellet of 4.0 µmol l–1 F-actin and 1.0 µmol l–1 Vma2pArch-MBP. Lanes 3 and 4 are the supernatant and pellet of 8.0 µmol l–1 F-actin and 1.0 µmol l–1 Vma2pArch-MBP supernatant.

 

Figure 3
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  		Fig. 3. Mutations in the profilin-like region of yeast B subunit do not affect assembly of bafilomycin-sensitive ATPase activity. (A) A yeast strain lacking yeast B subunit (VMA2{Delta}; lanes 1 and 4) was transformed with Vma2p (lanes 2 and 5) or Vma2pArch and separated by SDS-PAGE, blotted, and probed with monoclonal antibodies against yeast A subunit (top panel) or yeast B subunit (bottom panel). Lanes 1–3 are whole-cell blots; lanes 4–6 were immunoprecipitated with anti-B subunit and probed with anti-A subunit. The heavy chain (HC) of the immunoprecipitating antibody was detected in lanes 4–6. This experiment was repeated three times, the subunit A bands and heavy chain bands were analyzed by densitometry and the ratio of subunit A:heavy chain was determined. For wild-type cells the ratio was 0.94±0.02, for the Vma2pArch-transformed cells the ratio was 0.95±0.03. This indicates that very similar amounts of subunit A were immunopreciptated with anti-B antibody from VMA2{Delta} yeast transformed with wild-type yeast B subunit or Vma2pArch. BL, blank. (B) Vacuolar membranes were prepared for assay for bafilomycin-sensitive ATPase activity as described in Materials and methods. Samples of the vacuolar membranes from VMA2{Delta} yeast (lane 1), and VMA2{Delta} yeast transformed with yeast B subunit (lane 2) or with Vma2pArch plasmids (lane 3) were subjected to SDS-PAGE and immunoblotted to demonstrate the levels of subunit A and B associated with membranes. (C) ATPase assays were performed on the remainder of the membranes. Activities are expressed relative to vacuolar membranes isolated from the VMA2{Delta} strain transformed with Vma2p, which was defined as 100% and had a specific activity of 1.11 µmol l–1 ATP min–1 mg–1 protein in this experiment. Vacuolar membranes from the null yeast were significantly different from those of null yeast transformed with Vma2p (Student's t-test, *P<0.05). The membranes from the yeast transformed with Vma2pArch were also significantly different from those of the null yeast, but not significantly different from those of the yeast transformed with Vma2p.

 

Figure 4
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  		Fig. 4. Vma2pArch-containing yeast show increased sensitivity to cycloheximide and wortmannin. VMA2{Delta} yeast cells carrying no plasmid (Null) or transformed with yeast B subunit (WT), Vma2pPhe (Phe) or Vma2pArch (Arch) plasmids were grown to log phase, carefully counted so that each type of cell was at the same starting concentration, subjected to serial 10-fold dilutions, and then transferred to plates with control media (A) or media containing sub-lethal doses of various drugs (B) as indicated in Materials and methods for 3 days. Ct, camptothecin; Hu, hydroxyurea; Cx, cycloheximide; Wt, wortmannin; Sm, sulfometuron methyl; Fk, FK506.

 

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