Isabel Olivero

Regulation of β-exoglucanase activity production by Saccharomyces cerevisiae in batch and continuous culture

The rate of synthesis and secretion of exo-1–3-β-glucanase activity closely paralleled the specific rate of growth in exponentially growing Saccharomyces cerevisiae cells in batch culture. When the stationary phase was reached both synthesis and secretion stopped. No activity was synthesized when the cells were maintained in carbon sources that did not allow them to grow. Studies in continuous culture indicate a strong relationship between the synthesis of exoglucanase activity and the specific growth rate. These results are taken as evidence of an essential role of this activity during the yeast budding cycle.

Identification of low-dye-binding (ldb) mutants of Saccharomyces cerevisiae.

We have completed the identification of Saccharomyces cerevisiae genes that are defective in previously isolated ldb (low-dye-binding) mutants. This was done by complementation of the mutants phenotype with DNA fragments from a genomic library and by running standard tests of allelism with single-gene deletion mutants of similar phenotype. The results were as follows: LDB2 is allelic to ERD1; LDB4 to SPC72; LDB5 to RLR1; LDB6 to GON7/YJL184W; LDB7 to YBL006C; LDB9 to ELM1; LDB10 to CWH36; LDB11 to COG1; LDB12 to OCH1; LDB13 to VAN1; LDB14 to BUD32; and LDB15 to PHO85. Since the precise function of some of the genes is not known, these data may contribute to the functional characterization of the S. cerevisiae genome.

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.

Accumulation and secretion of exoglucanase activity in yeast secretory mutants

Representative conditional yeast secretory mutants, blocked in transport of secretory and plasma membrane proteins from the endoplasmic reticulum (sec 18), from the Golgi body (sec 7) and in transport of secretory vesicles (sec 1), accumulated exoglucanase, a constitutive yeast activity, when incubated at the restrictive temperature (37°C). Different proportions of the accumulated activity were released by mutant cells under permissive conditions. The presence or absence of cycloheximide during the secretion period made no differences in the results. More than 90% of the internal activity was bound to membrane in wild type cells. However, only the soluble pool underwent changes during the accumulation or secretion periods. The bulk of secretory invertase accumulated by sec 1 was also soluble. By contrast sec 7 and sec 18 accumulated membrane-bound as well as soluble invertase forms and both were secreted in similar proportions in each mutant. More than 90% of the accumulated invertase was secreted at the permissive temperature in sec 18 cells. That percentage was significantly lower for exoglucanase (<65%). Concomitantly, invertase accumulated by this mutant exited from the cells with a lower half time (t 1/2=150 min). These results may be interpreted assuming that exoglucanase is exported by a passive flow of the soluble pool.

The ldb1 mutant of Saccharomyces cerevisiae is defective in Pmr1p, the yeast secretory pathway/Golgi Ca2+/Mn2+-ATPase

The LDB1 gene of Saccharomyces cerevisiae was identified by complementation of the ldb1 mutant phenotype with a genomic library. We found that the ldb1 defect is complemented by PMR1 which codes for the yeast secretory pathway/Golgi Ca(2+)/Mn(2+)-ATPase. Besides that, the analysis of a null mutation of the PMR1 gene revealed a phenotype identical to that of ldb1 mutant. Thus, LDB1 must be considered a synonym of PMR1.

Two ionic forms of exoglucanase in yeast secretory mutants

Analysis of exoglucanase activity accumulated by sec mutants from Saccharomyces cerevisiae revealed the presence of two ionic forms of the major exoglucanase (exo II) secreted into the culture medium. From the accumulation pattern of representative sec mutants and the carbohydrate composition it appears that the less acidic form is converted into the more acidic one by addition of one phosphate to one of the oligosaccharide cores as the enzyme progresses through the secretory pathway. Exoglucanase I, the heavier isoenzyme, was not accumulated by the mutants. Accordingly, it should arise from exoglucanase II after the execution point of sec1 mutation.

The mnn2 mutant of Saccharomyces cerevisiae is affected in phosphorylation of N-linked oligosaccharides.

We studied the phosphorylation of the inner core region of N‐linked oligosaccharides in the mannan defective mutant Saccharomyces cerevisiae mnn2 which was described as unable to synthesize branches on the outer chain. We performed structural studies of the N‐oligosaccharides synthesized by the strains mnn2, mnn1mnn2mnn9 and mnn1mnn9ldb8, and the results are compared with previously published structural data of mnn1mnn2mnn10 and mnn1mnn9 [Hernández, L.M., Ballou, L., Alvarado, E., Tsai, P.‐K. and Ballou, C.E. (1989) J. Biol. Chem. 264, 13648–13659]. We conclude that the mnn2/ldb8 mutation is responsible for the inhibition of incorporation of phosphate to mannose A3 (see below), a particular phosphorylation site of the inner core, while phosphorylation at the other possible site (mannose C1) is allowed, although it is also reduced. *Phosphorylation sites in mnn1mnn9.

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.

Proteolytic processing of a secreted glycoprotein and O-glycosylation of mannoproteins are affected in the N-glycosylation mutant Saccharomyces cerevisiae ldb1

In a previous work [P.I. Mañas, I. Olivero, M. Avalos, L.M. Hernández, Glycobiology, 7 (1997) 487-497], we described the isolation and characterization of the Saccharomyces cerevisiae ldb1 mutant which is affected in several steps of the N-glycosylation of mannoproteins probably due to a malfunction of the Golgi apparatus. Here, we found that two further functions assigned to the Golgi cisternae are also affected in the mutant: proteolytic processing of a secreted protein and O-glycosylation. We found that around 70% of the exoglucanase activity that is secreted into the culture medium by ldb1 bears an extra tetrapeptide in its NH2-terminus due to incomplete proteolytic processing. The O-linked oligosaccharides from ldb1 mnn1 were indistinguishable from those synthesized by the parental strain mnn1. However, when the O-oligosaccharides from the wild type and ldb1 were compared, we found a significant decrease in the tetrasaccharide in the latter, as well as a concomitant increase in the disaccharide, suggesting a defect in the Kre2p/Mnt1p involved in the transfer of the third mannose of these residues.

Accumulation of exoglucanase activity in yeast secretory mutants blocked at the endoplasmic reticulum level

Saccharomyces cerevisiae HMSF‐176 (sec 18), a thermosensitive secretory mutant blocked at the endoplasmic reticulum (ER) level, drastically increased its osmotic sensitivity when grown at the restrictive temperature of 37°C in high glucose concentration. This fact led to the erroneous interpretation that glucanases were inactive when localized in the ER. The development of a suitable osmotic stabilizer now indicates that sec 18 accumulates exoglucanase activity. Another ER‐blocked mutant behaved in a similar way. All the accumulated exoglucanase was found in a soluble form. By contrast, a significant portion of the accumulated invertase remained in a membrane‐bound form.