Vacuole of yeast

Figure 2.10 NMR spectra of acid-soluble PolyPs extracted from vacuoles of yeast (Saccharomyces cerevisiae) (a) PolyP precipitated from the extract with Ba2+ at pH 8.2 (b) PolyP precipitated from the extract with Ba2+ at pH 4.5 1, middle phosphate groups 2, pre-terminal phosphate groups 3, terminal phosphate groups 4, y-phosphate groups of nucleoside triphosphates 5, Pj (Trilisenko et al., 2002).

First, the synthesis of ATP from PolyP has been observed in isolated vacuoles of yeast (Schabalin etal, 1977). However, the significance of this process needs further investigation. 

In the lower eukaryotes, cation sequestration and storage are observed in vacuoles. Vacuoles of yeast accumulate amino acids (Wiemken and Durr, 1974), K, Mg2+ and Mn2+ (Okorokov et al, 1980 Lichko et al, 1982) (Table 7.1), and Ca2+ (Ohsumi and Anraku, 1983 Dunn et al, 1994). PolyP, which is able to confine different cations in an osmotic inert form, was also found in these storage organelles (Indge, 1968a,b,c Westenberg et al, 1989). 

M. Diirr, K. Urech, T. Boiler, A. Wiemken, J. Schwencke and M. Nagy (1979). Sequestration of arginine by polyphosphate in vacuoles of yeast Saccharomyces cerevisiae. Arch. Microbiol., 121, 169-175. 

P pastoris has a unique advantage that it is capable of growing on minimal media to achieve a relatively high biomass (Cereghino and Cregg 2000). Furthermore, the excessive SAM can be sequestered in the vacuole of yeast cell, so yeast (such as P. pastoris and S. cerevisiae) can be regarded as an optimal host strain for SAM synthesis in vivo (Chan and Appling 2003 Shobayashi etal. 2007). Many reports have been published to document improvements of SAM production by P. pastoris (Li et al. 2002 Yu et al. 2003). 

The lysosome is an acidic organelle containing many hydrolases and can degrade most biological macromolecules (Kornfeld and Mellman, 1989). The vacuole in yeast and plants is thought to be its functional relative. Both of them are bounded by a single membrane and have... 

An H+ electrochemical gradient (ApH+) provides the energy required for active transport of all classical neurotransmitters into synaptic vesicles. The Mg2+-dependent vacuolar-type H+-ATPase (V-ATPase) that produces this gradient resides on internal membranes of the secretory pathway, in particular endosomes and lysosomes (vacuole in yeast) as well as secretory vesicles (Figure 3). In terms of both structure and function, this pump resembles the F-type ATPases of bacteria, mitochondria and chloroplasts, and differs from the P-type ATPases expressed at the plasma membrane of mammalian cells (e.g., the Na+/K+-, gastric H+/K+-and muscle Ca2+-ATPases) (Forgac, 1989 Nelson, 1992). The vacuolar and F0F1... 

Vacuoles also contain an important phosphorus reserve in yeast and fungi (Indge, 1968a,b,c Urech et al, 1978 Cramer and Daves, 1984). Under phosphate overplus, the content of PolyP in vacuoles of Saccharomyces carlsbergensis grew dramatically (Lichko et al., 1982). Some mutants of S. cerevisiae having no vacuoles possess low levels of PolyP and are unable to grow on a medium without P (Shirahama et al., 1996). 

The accumulation of Mn2+ in the vacuoles of Saccharomyces carlbergensis (Lichko et al., 1982) correlated well with the increase in PolyP content (Table 7.2). During the accumulation of Mn2+ by S. carlsbergensis, both of the PolyP and Mn2+ contents increased simultaneously. This accumulation took place even when the incubation medium contained no Pi and was accompanied by a simultaneous decrease of Pi content in the vacuoles. This complex-forming function of PolyP may be very important for the yeast cell, since under a short-term phosphate starvation in the presence of metal cations in the medium the vacuolar PolyP content slightly decreases (Table 7.3) (Lichko et al., 1982). A stable Pi content in the cytosol under the above conditions is maintained mainly due to a decrease in the vacuolar P pool but not in the vacuolar PolyP pool. It is probable that the ability of fungi to accumulate large amounts of heavy metals is connected with the PolyP pools, especially in vacuoles. 

While PolyPs are localized in different compartments of the yeast cell, it is important to determine the effects of P starvation and P, overplus on PolyPs in organelles. The content of PolyP in vacuoles of the yeast S. cerevisiae was 15 % of the total cellular PolyP. Over 80 % of vacuolar PolyPs were represented by the acid-soluble fraction. It was established by 31P NMR spectroscopic studies that the polymeric degrees ( ) of two subfractions obtained by precipitation with Ba2+ ions in succession at pH 4.5 and 8.2 were approximately 20 5 and 5 2 residues of orthophosphoric acid, respectively. Under the deficit of phosphate (P ) in the culture medium, the PolyP content in vacuoles decreased sevenfold at the same drastic reduction of its content in the cell. Unlike the intact yeast cells where PolyP overcompensation is observed after their transfer from phosphate-free to phosphate-containing medium, the vacuoles do not show this effect (Table 8.2). The data obtained indicate the occurrence of special regulatory mechanisms of PolyP synthesis in vacuoles differing from those in the whole cell. 

Table 8.2 PolyP content (mg of Pi per g of dry cell biomass) in the cells, spheroplasts and vacuoles of S. cerevisiae. The yeast was grown for 4 h in a medium with 9 mM P (+P), then for 7 h in Pi-free medium (—P) and finally for 2 h in a medium with 9 mM P (+P, phosphate overplus) (Trilisenko et al., 2002).

L. Lichko and L. Okorokov (1991). Purification and some properties of membrane-bound and soluble pyrophosphatases of yeast vacuoles. Yeast, 7, 805-812. 

In this experiment, we will culture fission yeast cells from the S. pombe strain in the presence of Cd2+. In response, the yeast will express PCS that will begin PC synthesis and uptake of the Cd2+ into the vacuole of the cell. We will then extract the Cd-PC2 complexes and analyze the Cd2+ content by atomic absorption spectroscopy. Appendix 5 describes the apparatus and gives reagents needed for yeast culture. 

Early evidence that membranes can be delivered to the lumen of compartments came from electron micrographs showing membrane vesicles and fragments of membranes within endosomes and lysosomes (see Figure 5-20c). Parallel experiments in yeast revealed that endocytosed receptor proteins targeted to the vacuole (the yeast organelle equivalent to the lysosome) were primarily associated with membrane fragments and small vesicles within the interior of the vacuole rather than with the vacuole surface membrane. 

Yeast, which, like microalgae, produce PCs in response to high Cd concentrations transport the Cd-PC complex from the cytoplasm to the vacuole via a transport molecule in the vacuolar membrane [41]. In marine diatoms, radiotracer studies have shown that Cd is exported at high external Cd concentration (Cd = 5 nM [42,43]). It seems that this export system may be similar to that of yeast and export the Cd-PC complex outside of the cell. 

Fig. 2.7 Germ tube formation by Candida albicans. For simplicity the diagram merely illustrates the nuclear content of the parental yeast cell and the developing germ tube. In real life there is a complex rearrangement of cytoplasmic constituents which results in all parts except the apex becoming highly vacuolated.

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