Vacuolar morphology and cell cycle distribution are modified by leucine limitation in auxotrophic Saccharomyces cerevisiae

Yeast vacuoles are highly dynamic and flexible organelles. In a previous pa per, we have shown that subtle, often unrecognised amino acid limitations l ead to much lower final cell densities in cultures of different commonly us ed auxotrophic Saccharomyces cerevisiae strains (Cakar et al., Biotechnol. Lett. 21 (1999) 611). Here, we demonstrate for two of these strains, CEN.PK 113.6B and CBS7752, that such subtle leucine limitations also affect the n umber and morphology of vacuoles, and that these changes are correlated wit h the cell cycle in batch cultures in a similar way as is known from synchr onized cultures. Morphological aspects were studied by electron microscopy, using advanced high pressure freezing/freeze-substitution techniques for s ample preparation that so far have been barely successful in yeast. Cells o f leucine-limited cultures had single, large vacuoles with a hexagonal tono plast pattern and were partially arrested in G1 phase. To relieve leucine-l imitation, additional leucine was supplied extracellularly via the medium o r intracellularly via enhanced leucine biosynthesis due to plasmid-based ex pression of a leucine marker gene. Such cultures reached more than two-fold higher final optical densities in stationary phase. Cells in later growth phase were characterized by fragmented vacuoles lacking any tonoplast patte rn and by a smaller proportion of cells in G1 phase. These drastic effects of subtle leucine limitation on cell physiology, vacuolar morphology and ce ll cycle distribution present a note of caution for morphological and cell cycle studies in yeast. (C) 2000 Editions scientifiques et medicales Elsevi er SAS.