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Book of Abstracts: Albany 2007

category image Albany 2007
Conversation 15
June 19-23 2007

Functional Forms of the Yeast Plasma Membrane H+-ATPase Carrying Multiple Alanine Substitutions of Cysteine Residues

Pma1 H+-ATPase, which is located in the plasma membrane of yeast (Saccharomyces cerevisiae) and encoded by the PMA1 gene (1, 2), belongs to the P2 subfamily of cation-transporting pumps, widely spread among pro- and eukaryotes. This H+ pump generates a transmembrane electrochemical proton gradient (ΔμH+) that provides energy for multiple secondary solute transport systems. It contains nine cysteines, three of them (Cys-148, 312, and 867) located in transmembrane segments that anchor the ATPase in the lipid bilayer and the rest (Cys-221, 376, 409, 472, 532, and 569) located in cytosolic parts of the molecule (Table I).



Because cysteine residues are highly reactive, it would be useful to introduce them into desired positions in order to determine accessibility to SH reagents, measure distances in the 3D structure, and monitor conformational changes during the reaction cycle. First, however, one would need to eliminate as many as possible of the existing cysteines in order to minimize the background in labeling experiments. In previous work (3), we found that replacement of the nine existing cysteines one at a time led to little or no reduction in ATPase activity, indicating that no single Cys residue is essential for function. On the other hand, substitution of all of them led to the impairment of biogenesis and loss of enzyme activity (3). The present study has used site-directed mutagenesis combined with other molecular biological and biochemical methods to define different combinations of minimal cysteine content that are consistent with ATPase function.

As reported previously, data on single and multiple substitutions of Cys residues by Ala pointed to an important role of Cys-409: presence of this single residue allowed the enzyme to be expressed with detectable activity (Table II, Ref. 3). This residue also appears to play an important role in inhibition of the ATPase by N-ethylmaleimide (NEM) and fluoresceine 5'-isothiocyanate (FITC), since substitution of Cys-409 by Ala or Ser enhanced the reaction of the ATPase with NEM at Cys-532 and with FITC at Lys-474 (5). Another important residue is Cys-472 (Table II), especially in combination with Cys-409. Indeed, compared to the wild type, replacement of both Cys-409 and Cys-472 by Ala led to 2.5 fold decrease of ATPase activity (3), while an enzyme with only these two cysteines (C409/472) had half of the normal ATPase activity (Table II, Ref. 3).

We obtained and examined additional two- and three-cysteine combinations (Table I) aiming at improving the functional activity of the ATPase by comparison with the previously obtained C409/472 mutant (Table II; Ref. 3). Among seven additional two-cysteine mutants, two (C312/409 and C376/472) appeared to be close to or slightly better than C409/472. However, the activity of these two-cysteine mutants was not high enough to use them for labeling experiments. It is worth mentioning that combining Cys-409 with one of two NEM-reactive Cys residues (Cys-221 or Cys-532; Ref. 5), resulted in a significant drop of ATPase activity, lowering it to that of the one-cysteine C409 mutant (Table II; Ref. 3).

Expression and activity of the three-cysteine forms were higher: out of five mutant ATPases, two of them (C312/409/472 and C376/409/472) were similar to the wild type control, and one (C148/409/472) had activity slightly lower than the wild type control. The three-cysteine C221/409/532 mutant had activity identical to the one-cysteine C409 ATPase (Table II).


Thus, among fourteen multiple cysteine mutants, three (C148/409/472, C312/409/472, and C376/409/472) could be used for future experiments involving labeling the ATPase with radioactive, fluorescent, or spin-labeled SH reagents. An additional advantage of these mutants is that the remaining Cys residues reside in different parts of the molecule, which makes them accessible to one (hydrophobic or permeant) and not accessible to another (hydrophilic or impermeant) SH reagents. Therefore, they can be used as is or by introducing new Cys residues at points of interest.

Acknowledgement

This research was supported in part by Research Grant GM15761 from the NIGMS, National Institute of Health, the Russian Foundation for Basic Research grant 07-04-00419-a, and the Support Fund of Leading Scientific Schools of Russia NS-4318.2006.4.

References and Footnotes
  1. Ulaszewski, S., Grenson, M., Goffeau, A. Eur J Biochem 130, 235-239 (1983).
  2. Serrano, R., Kielland-Brandt, M. C., Fink, G. R. Nature 319, 689-693 (1986).
  3. Petrov, V. V., Slayman, C. W. J Biol Chem 270, 28535-28540 (1995).
  4. Nakamoto, R. K., Rao, R., Slayman, C. W. J Biol Chem 266, 7940-7949 (1991).
  5. Petrov, V. V., Pardo, J. P., Slayman, C. W. J Biol Chem 272, 1688-1693 (1997).

Valery V. Petrov1, 2, *
Carolyn W. Slayman1

1Department of Genetics
Yale University School of Medicine
333 Cedar Str.
New Haven, CT 06510, USA
2Laboratory of Regulation of Biochemical Processes
Institute of Biochemistry and Physiology of Microorganisms
Russian Academy of Sciences
142290 Pushchino, Russia

*Phone: +7 496 773 0548
Fax: +7 495 956 3370
Email: vpetrov06@mail.ru