Dependence of Saccharomyces cerevisiae Golgi functions on V-ATPase activity.

The V-ATPase of Saccharomyces cerevisiae is an ATP-dependent proton pump responsible for acidification of the vacuole and other internal compartments including the whole secretory pathway. We have studied the behavior of several glycoprotein processing reactions occurring in different Golgi compartments of representative vmaΔ mutants. We found that outer chain initiation is not altered in the mutants while mannosylphosphate transfer, α(1,3)-linked mannoses addition, and α factor maturation seem to be affected. The results suggest a gradation in the dependence of Golgi functions on V-ATPase activity, from early Golgi (unaffected) to late Golgi (significantly reduced). These findings are in agreement with the internal pH of Golgi cisternae measured in mammalian cells, which is more acidic in the late region. The mutant defects can be partially restored by buffering the external medium to pH 6.0, which supports the existence of a mechanism that, in the absence of a functional V-ATPase, could contribute to pH regulation at least in the late Golgi.

Standard YPD, even supplemented with extra nutrients, does not always compensate growth defects of Saccharomyces cerevisiae auxotrophic strains.

Conventional complex media are routinely used to grow auxotrophic strains under the assumption that they can compensate the latter's nutritional deficiencies. We here demonstrate that this is not always true. This study compares the growth parameters of Saccharomyces cerevisiae (S288C) and its derived auxotrophic strains FY1679-14C and BY4741 in synthetic minimal medium (SD), standard YPD medium from two of the most commonly used suppliers, or modified YPD medium. Maximum specific growth rates of auxotrophic strains were slightly lower than the prototrophic case in all growth conditions tested. Also, the biomass production of auxotrophic strains in synthetic medium was slightly less than the prototrophic case. However in both of the two standard YPD media used, the biomass production of both auxotrophic strains was markedly lower than that of the prototrophic one. The extent of the differences depended on the medium used. Indeed in one of the two YPD media, the lower biomass production of auxotrophic strains was evident even at the diauxic shift. Uracil seems to be the main limiting growth factor for both auxotrophic strains growing in the two standard YPD medium tested. No YPD media or specific supplement was able to compensate for the effect of the auxotrophic mutations in the multiple auxotrophic marker strain BY4741. The fact that auxotrophic strains grew poorly on YPD when compared to their prototrophic counterpart indicates that standard YPD medium is not sufficient to overcome the effect of auxotrophic mutations.