Accumulation of ergosterol during the cell cycle of the budding yeast Kluyveromyces fragilis

Abstract

It has long been known that ergosterol is the main sterol in yeast cells. Recent studies involving the use of gas-liquid chromatography and mass spectrometry have revealed the presence of other sterols in significant quantities in flaccharomyees cerevisiae (LONOLEY et al. 1968, HUNTER and ROSE 1972, BARTON et al. 1972) but the present authors have found that in Kluyveromyces fragilis only ergosterol is detectable (PENMAN and DUFFUS 1974). Because of the relative paucity of knowledge concerning the flux of lipid components during the cell cycle, it seemed of interest to examine the accumulation of this sterol fraction during the cell cycle in K . fra- gilis.Diethyl ether was redistilled before use and stored dry. Cyclohexane was obtained from British DrugHouses Ltd.Stock cultures of Kluyveromyces fragilis NCYC 100 were maintained on 6% OXOID Malt Extract Agar and batch cultures grown up in 2% OXOID Malt Extract Broth (MEB) at 30 "C in an orbital incubator operating a t 160 rpm.Synchronous cultures were prepared using the selection method of MITCHISON and VINCENT (1966). Where it was desirable to obtain larger numbers of synchronously dividing cells, two or more identical gradients were used and cells removed from exactly the same point in each gradient. After re-inoculation into fresh growth medium, the yeasts were allowed to grow for one hour before sampling, after which time samples were removed at 20 min intervals and cell numbers determined using a haemacytometer (DUFFUS and PENMAN 1973). These samples were frozen rapidly by immersion in a solid CO,/ethanol bath and stored at -18 "C. For analysis, the samples were thawed and the yeasts collected by centrifuging a t 0 "C. Each pellet was washed twice with distilled water and saponified under reflux for two hours using the method described by HUNTER and ROSE (1972). After the addition of 10 ml distilled water t o each sample and acidification t o p H 1.0 with N HCI, the saponified mixtures were extracted 3 times with diethyl ether (5 ml) portions. The ethereal extracts were dried over anhydrous Na,SO, and drying was completed under vacuum. The dried extracts were taken up in 10 ml volumes of cyclohexane and sterol contents estimated as ergosterol by the spectrophotometric method of S m w and JEFFERIES (1953). Extinction measurements were taken at 282 nm, 294nm, 272 nm, and 310nm against a cyclohexane blank and substituted in the equations of SHAW and JEFFEKIES (1953). Fig. 1 shows the total extracted ergosterol from a typical synchronous culture. Whereas the cell numbers increase in a stepwise fashion, the ergosterol increases in a continuous manner. The plot of the logarithm of the total sterol is a straight line, showing that the sterol increase is exponential. I n five separate experiments, the level of ergosterol increased through successive cell cycles in such a way that the cells of each succeeding cycle contained more ergosterol at any given time than a t the corresponding time in previous cycles.The surface area of a growing cell doubles during an ideal...