Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Role of M5-M6 loop in the biogenesis and function of the yeast Pma1 H+-ATPase
Yeast Pma1 Н+-АТРase belongs to P2-type ATPases which couple ATP hydrolysis to transport of cations across biomembranes where they are embedded by 10 membrane segments. The determinants of cation specificity and stoichiometry lie in M4, M5, M6, and M8 segments; point mutations in these segments affect normal functioning and biogenesis of the enzyme and change both stoichiometry (1-3) and specificity (4). During the reaction cycle P2-ATPases undergo significant conformational changes: M1-M6 segments bend, unwind partially and even shift normal to the membrane (5). Ala substitutions of the residues in the M6 N-terminal half interfered markedly with the Pma1 functioning and biogenesis, or both (3). The results described here extend a systematic study of the yeast H+-ATPase by focusing on the extracytoplasmic loop between M5 and M6 segments of this enzyme, searching for residues that may play a role in H+ transport or any other aspect of the enzyme functioning and biogenesis. To explore role of this loop in the structure-function relationship of the S. cerevisiae Pma1 АТРase, Ala-scanning mutagenesis was used. The loop consists of 7 residues: 714-DNSLDID. L717 is the most conservative among them. Accordingly, only L717A led to a complete block in membrane trafficking that prevented the ATPase from reaching secretory vesicles (SV) which points to a severe defect in protein folding, causing the abnormal ATPase to be retained in the endoplasmic reticulum (ER). Mutation D714A was expressed poorly in SV (Fig. 1), displaying very low ATPase activity with no detectable H+ pumping. The remaining 5 mutations were expressed at 34 to 94% with ATPase activities of 35 to 101% of the WT level. Given the known contribution of M5 and M6 segments to the transport pathway of P2-ATPases and their mobility during reaction cycle, it was of particular interest to ask whether any of the mutations in the M5-M6 loop affected H+ pumping. For most of the mutants, the coupling ratio was close to that of the WT (1.00). Only I719A mutant gave the ratio significantly lower (0.29), pointing to a partial uncoupling between ATP hydrolysis and H+ transport. Thus, 3 of 7 residues in M5-M6 loop seem to be important for the proper function and biogenesis of the yeast Pma1 H+-ATPase. L717 is located in the middle of the loop; since the enzyme reaction cycle is accompanied by significant conformational changes, substitution of this residue with a smaller Ala may strongly affect the mobility of M5 and M6 segments causing misfolding and retaining of impaired enzyme in ER. D714 is also important for structure and, especially, functioning of the enzyme; it probably plays a role similar to D739 in M6 which neutralizes neighboring positive charge and, thus, stabilizing the protein (3). Finally, I719 replacement with Ala caused significant uncoupling between ATP hydrolysis and H+ transport.
Author is grateful to scientific adviser of this project Prof. C. W. Slayman (Yale School of Medicine).
Valery V. Petrov
Institute of Biochemistry and Physiology of Microorganisms, RAS, 142290 Pushchino, Russia
Fig. 1. Effect of Ala substitutions of the residues in the M5-M6 loop of the Pma1 ATPase on the enzyme expression (left columns) and activity (right columns), %.