Fungicides and sterol-deficient mutants of Ustilago maydis: plasma membrane physico-chemical characteristics do not explain growth inhibition

Abstract

Plasma membrane vesicles from erg1 I and erg2 sterol-deficient mutants and from wild-type Ustilago maydis sporidia treated with and without inhibitors of sterol 14a-demethylase or sterol A8-A7 isomerase (triadimenol and fenpropimorph fungicides, respectively) were purified by aqueous two-phase partitioning. Changes in plasma membrane lipid composition were mostly restricted to sterols and complex lipid-bound fatty acids (CLB fatty acids). There was a greater accumulation of abnormal sterols (14a-methyl-or A8-unsaturated sterols) in plasma membranes from sterol-def icient mutants than from those treated with their fungicide counterparts. However, greater growth inhibition was observed on fungicide-treated wild-type than on mutants. Changes in CLB fatty acids were restricted to alterations in the relative proportion of linoleic acid (18:2) with respect to oleic acid (18: 1). The 18:2 to 18:l ratio found in CLB fatty acids in plasma membranes could be correlated to rates of sporidial growth but not to accumulation of a particular abnormal sterol or to the extent of sterol replacement. Plasma membrane permeability to protons was increased moderately in the mutants only. No changes were observed in plasma membrane fluidity. Plasma membrane H+-ATPase activity was increased up to twofold in those cases with lower growth rate. It was concluded that fungiciddnduced growth inhibition in U. maydis was not due to accumulation of abnormal sterols in plasma membranes but probably due to intracellular ATP depletion by the H+-ATPase and that changes in 18:2 to 18: 1 ratio in CLB fatty acids were not directly dependent on the plasma membrane physical state or lipid composition but were possibly part of a stress adaptation mechanism.

In vivo activation of plasma membrane H+‐ATPase hydrolytic activity by complex lipid‐bound unsaturated fatty acids in Ustilago maydis

“…It could be argued that this activation may be due to other causes such as increased polypeptide amounts in membrane or changes in fluidity. We have shown previously that these two factors remain largely unchanged in the same conditions used in this study [18,21].…”

“…A␣close correlation ( r =0.98) was observed, and this was independent of the kind of genetic lesion or inhibitor used, suggesting that this activating effect was indeed caused by the fatty acid/lipid environment of the ATPase. It must be noted that triadimenol and fempropimorph have no effect on ATPase activity in these conditions [21]. The 18 : 2/18 : 1 ratio in untreated wild‐type was close to unity.…”

“…In our system, the modification of the fatty acid moieties was provoked by the presence of abnormal sterols plus, in the case of EBI‐treated sporidia, other collateral effects of these compounds [33]. In U. maydis , changes in the plasma membrane 18 : 2/18 : 1 ratio followed the inhibition of growth rate, which was influenced not only by the biosynthetic point affected but also by the method used [21]. These changes correlated directly with increases in the H + ‐ATPase specific activity which could, in its turn, be mimicked by altering the 18 : 2/18 : 1 ratio of isolated plasma membrane vesicles in vitro .…”

“…Methyl heptadecanoate was added as an internal standard and the plasma membrane lipids were extracted as described [21]. CLB‐fatty acids were quantified by GC analysis.…”

Effects of Abnormal-Sterol Accumulation on Ustilago maydis Plasma Membrane H + -ATPase Stoichiometry and Polypeptide Pattern

“…To date, no report has dealt with the effects that abnormal-sterol accumulation may have on the fungal plasma membrane H ϩ -ATPase, although the lipid composition of the plasma membrane is thought to be of great importance in membrane-bound enzyme activity. In another publication, we showed that it is useful to make a comparison between the effects of a fungicide on abnormal-sterol composition and those found in a mutant with a genetic block in the sterol biosynthesis pathway at the point where the fungicide is presumed to act (6). These comparisons showed that the fungicides produced effects, similar to stress adaptation processes, which were not found in the mutants (6).…”