Book of Abstracts: Albany 2009

category image Albany 2009
Conversation 16
June 16-20 2009
© Adenine Press (2008)

Heat Shock Affects Functioning of the Yeast Plasma-membrane Pma1 H+-ATPase

The yeast Pma1 H+-ATPase belongs to the subfamily of P2-type ATPases, a part of large and wide-spread family of P-type ATPases found throughout pro- and eukaryotes which also includes mammalian K+, Na+, H+, K+, and Ca2+-ATPases (1). These pumps couple ATP hydrolysis to transport of different cations across plasma membrane thus generating electrochemical gradient of ions and maintaining cell homeostasis. Structurally, the P2-ATPases share a common topology in which a large cytosolic catalytic domain is connected with a small extracellular part through 10 hydrophobic segments embedding the protein in the lipid bilayer. This transmembrane domain forms translocation pathway and contains sites for cation binding. Cryoelectron microscopic studies that had defined the number of transmembrane α-helices and site-directed mutagenesis had clearly implicated M4, M5, M6, and M8 in high-affinity cation binding (2). Of these segments, M8 has been less characterized and its role is not yet clear. Recently we have described a set of mutants made by Ala-scanning mutagenesis to examine the functional role of amino acid residues throughout M8 of the yeast Pma1 H+-ATPase (3). Initially, these mutant proteins were expressed from a centromeric plasmid in the yeast strain SY4 where secretory vesicles containing ATPase become arrested due to a temperature-sensitive block preventing vesicle fusion with the plasma membrane under heat shock. At this step, we found that 4 of 21 alanine substitution (I794A, F796A, Q798A, and I799A) could not overcome quality control points under stress condition and appeared to be retained in an early stage of biogenesis due to impaired folding (3). To understand better the role of these residues, these four mutations were integrated into the chromosomal copy of PMA1 gene. Two alleles (Q798A and I799A) were unable to support growth at non-restrictive temperatures, 23°C and 30°C (3), while I794A and F796A strains grew slower than the wild-type (WT) even at 30°C. These mutants, especially I794A, showed signs of temperature sensitivity.

The expression of the ATPase gene (PMA1) is regulated by glucose (4) and the enzyme itself shows the phenomenon known as glucose activation (5): the Pma1 ATPase isolated from glucose-metabolized and starved cells has different activity and kinetic parameters. To study further the effect of these substitutions on the ATPase and influence of heat shock on the enzyme function and regulation, we isolated plasma membranes from cells which were starved and re-fed at permissive (30°C) and restrictive (37°C) temperatures. WT tolerated heat shock well showing just slight reduction in growth and the amount of ATPase protein. I794A strain showed two-fold reduction in the amount of the mutant ATPase to that seen in WT even during growth at 30°C. The difference became more profound at 37°C: the amount of the I794A enzyme from metabolizing cells was about one third of the control and in starved cells heat shock led to almost eight-fold drop suggesting that heat shock under starved conditions decreases stability of the I794A enzyme. The expression of the F796A ATPase was insignificantly affected by the cultivation temperature. Influence of heat shock on activity of ATPase and its activation by glucose was more noticeable both in the WT and mutant strains. For WT, activity dropped three fold in glucose-starved and two fold in glucose-metabolizing membranes compared with that in the membranes isolated from cells grown at 30°C. The changes in the mutant activities were more visible. Under glucose-metabolizing conditions the activity lowered by a half of the control for I794A and by a quarter of that for F796A. The ability of the F796A ATPase to be activated by glucose was significantly impaired, while for I794A it was almost abolished. Cultivation at 37°C caused a substantial decrease of the WT ATPase activity compared with the enzyme activity during growth at 30°C: to almost one third in the membranes isolated from starved and to a half in those isolated from metabolizing cells. For the I794A mutant, increasing temperature to 37°C also led to decrease of specific ATPase activity; however, the effect of high temperature was less dramatic. F796A mutant was less affected at 37°C. At the same time, the apparent ratio of the ATPase activation by glucose at 37oC was higher for the WT and, to a lesser degree, for the F796A mutant.

Fig. 1 shows a 3D model of the membrane domain of Pma1 H+-ATPase, built using the E1Ca structure of SERCA1a Ca2+-ATPase as a template (3). In this view, M8 is surrounded by five other transmembrane helices, with the M8 residues important for folding and biogenesis reaching out towards M4-M6 (I799), in between M6 and M9 (F796), M7 and M10 (I794 and Q798). Since Q798 is in close proximity to M7 and M10 and I799 is facing transport pathway formed by M4, M5, M6, and M8, these residues seem well positioned to play a structural and functional role by contributing to the proper assembly of helices within the M domain. F796 seems to be less important. I794 occupies intermediate position: it is important for the functioning and regulation of the enzyme, especially under stress conditions.

Figure. 1: Homology model of the yeast Pma1 H+-ATPase showing membrane domain from the extracytoplasmic surface of the membrane. Numbers indicate transmembrane segments 1 to 10.


The author is grateful to Prof. C. W. Slayman (Yale University) for support and scientific advising. This study was supported in part by the RFFI grant 07-04-00419, and Grant of the President of Russian Federation for the Leading Scientific Schools SS-1004.2008.4.

References and Footnotes
  1. Lutsenko, S., and Kaplan, J. H. Biochemistry 34, 15607-15613 (1995).
  2. Toyoshima, C., and Inesi, G. Ann Rev Biochem 73, 269-292 (2004).
  3. Guerra, G., Petrov, V.V., Allen, K.E., Miranda, M., Pardo, J.P., and Slayman, C.W. Biochim Biophys Acta 1768, 2383-2392 (2007).
  4. Rao, R., Drummond-Barbosa, D., and Slayman, C. W. Yeast 9, 1075-1084 (1993) ).
  5. Serrano, R. FEBS Lett 156, 11-14 (1983).

Valery V. Petrov

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