Effect of Polygodial on the Mitochondrial ATPase of Saccharomyces cerevisiae

Abstract

The fungicidal mechanism of a naturally occurring sesquiterpene dialdehyde, polygodial, was investigated in Saccharomyces cerevisiae. In an acidification assay, polygodial completely suppressed the glucose-induced decrease in external pH at 3.13 g/ml, the same as the fungicidal concentration. Acidification occurs primarily through the proton-pumping action of the plasma membrane ATPase, Pma1p. Surprisingly, this ATPase was not directly inhibited by polygodial. In contrast, the two other membrane-bound ATPases in yeast were found to be susceptible to the compound. The mitochondrial ATPase was inhibited by polygodial in a dose-dependent manner at concentrations similar to the fungicidal concentration, whereas the vacuolar ATPase was only slightly inhibited. Cytoplasmic petite mutants, which lack mitochondrial DNA and are respiration deficient, were significantly less susceptible to polygodial than the wild type, as was shown in time-kill curves. A pet9 mutant which lacks a functional ADP-ATP translocator and is therefore respiration dependent was rapidly inhibited by polygodial. The results of these susceptibility assays link enzyme inhibition to physiological effect. Previous studies have reported that plasma membrane disruption is the mechanism of polygodial-induced cell death; however, these results support a more complex picture of its effect. A major target of polygodial in yeast is mitochondrial ATP synthase. Reduction of the ATP supply leads to a suppression of Pma1 ATPase activity and impairs adaptive responses to other facets of polygodial's cellular inhibition.Systemic fungal infections are increasingly important causes of high morbidity and mortality (9, 18). Fungi are significantly affecting the growing population of patients with impaired immune systems due to AIDS, cancer chemotherapy, or immunosuppressive drugs. In addition to life-threatening systemic infection, superficial mycoses can clinically present as persistent infections that require continual treatment. Oropharyngeal and esophageal candidiasis occurs in over 70% of people with AIDS (46). With the increasing incidence of both systemic and superficial mycoses, it is critical that we develop new antifungal agents. Additionally, antifungal drug resistance has become an important problem and even further intensifies the need for new compounds (42, 54). Therefore, identifying promising cellular targets and understanding their physiological roles and basic biochemistry are critical for successful antifungal development (21,36).Polygodial (Fig. 1), a sesquiterpene dialdehyde, was originally isolated from the plant Polygonum hydropiper (3) and subsequently from Warburgia ugandensis and Warburgia stuhlmannii exhibiting insect antifeedant activity (28) and antimicrobial activity (48), as well as from Pseudowintera colorata exhibiting antimicrobial activity (34). Polygodial is a component of the "hot taste" in peppery spices of traditional Japanese cuisine (26). Unlike many other antifungal agents, polygodial has fungicidal activity against yeasts and...

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

“…Our activity assay and the use of inhibitors was based on (Serrano 1978;Clelland and Saleuddin 2000;Lunde and Kubo 2000;Padilla-Lopez and Pearce 2006), with modifications, as follows. The assay mixture contained the activity buffer (50 mM MES/TRIS, 5 mM MgCl 2 , pH 7.0), 5 mM sodium azide (to inhibit mitochondrial ATPase), 0.2 mM ammonium molybdate (to inhibit acid phosphatases), 100 μM sodium orthovanadate (to inhibit plasma membrane proton-ATPase).…”

“…Since the substrate of the reaction is MgATP and MgCl 2 is in excess to Na 2 ATP, the substrate concentration was close to 2 mM (at least in the beginning of the reaction). Several inhibitors that are known to inhibit other ATPases (Serrano 1978;Clelland and Saleuddin 2000;Lunde and Kubo 2000;Padilla-Lopez and Pearce 2006) were tested. These 'other' inhibitors removed only about 20% of the total ATPase activity, even at relatively high inhibitor concentration.…”

Alternative Respiratory Pathway under Drought is Partially Mediated by Hydrogen Peroxide and Contributes to Antioxidant Protection in Wheat Leaves

“…The conductivity was measured again. The conductivity was assessed by measuring 260-nmabsorbance released to the medium (Lunde and Kubo, 2000). Electrolyte leakage was calculated as the ratio of the value of conductivity measured.…”

Chemical composition and antifungal activity of essential oil ofHyssopus officinalisL. from Bulgaria against clinical isolates ofCandidaspecies

“…The external medium acidification after addition of glucose in the presence of hyssop oil was performed as described by Lunde and Kubo [44] with minor modifications. Amounts of essential oil stock solution were added to aliquots of yeast working suspension (2 £ 10 6 À4 £ 10 6 cfu¢mL ¡1 ) to reach the desired final oil concentrations.…”