The inability of ��Klpgs1 cells to grow on respiratory carbon sources and the decreased rate of multiplication of ��Klcls1 cells on glycerol were in correlation with oxygen consumption (Table MLN0128 research buy
2). The ��Klpgs1 mutant strain displayed total inability to respire ? its oxygen consumption was <1% of the parental strain. Only a slight decrease in oxygen consumption (80% that of the parental strain) was measured in ��Klcls1. The deletion of the KlCLS1 gene in the wild type led to a more severe decrease of oxygen consumption: about 50% that of the parental strain (Table S2). To determine whether KlPGS1 gene expression will be able to restore the ability of cells to grow on a respiratory substrate, the pCXJ19KGAL1PGS1 plasmid was transformed into the ��Klpgs1 mutant strain. The expression of the KlPGS1 gene was verified by Northern blot (Fig. 2). Despite the fact that the KlPGS1 gene was expressed, transformed cells were not able to grow on galactose. It is known that the growth of K. lactis cells on galactose requires oxidative phosphorylation due to the fact that this yeast species undergo a Kluyver effect, i.e. they are unable to assimilate some carbon sources only under respiring conditions (Entian & Barnett, 1983). It has been published that, on some genetic backgrounds, the S. cerevisiae��PGS1 strain is petite lethal, i.e it is unable to survive the loss of mtDNA (Dzugasova & Subik, 2005). Moreover, it was also shown recently that C. glabrata��PGS1 cells were unable to grow in the presence of ethidium bromide (Batova et al., <a href="http://www.selleckchem.com/products/OSI-906.html
">Linsitinib chemical structure 2008). To determine whether ��Klpgs1 is able to survive ethidium bromide mutagenesis, cells of the parental mgi1-1 strain, as well as ��Klpgs1 and ��Klcls1 strains, were tested for growth plated on yeast�Cpeptone�Cdextrose (YPD) plates with ethidium bromide. Colonies were tested for the presence of mtDNA. We found that not only parental mgi1-1 and ��Klcls1 strains (data not shown) but also the ��Klpgs1 strain was able to survive the loss of mtDNA (Fig. 3). In order to determine the influence of KlPGS1 and KlCLS1 gene disruption on the level of mitochondrial proteins, mitochondria isolated from SB 203580
mutant and parental strains were tested by Western blot analysis for some outer membrane, inner membrane and intermembrane proteins. In the ��Klpgs1 strain, only the amount of intermembrane space protein l-lactate cytochrome c oxidoreductase (cytochrome b2) (Fig. 4a, lanes 1 and 3) was the same as in the parental strain. In this strain, we observed a slight decrease in the amount of porin (Fig. 4a, lanes 1 and 3), but found that all investigated integral, as well as peripheral, proteins of the inner mitochondrial membrane (ADP/ATP carrier, cytochrome c and �� subunit of F1 ATPase,) were present at reduced levels when compared with the parental strain (Fig. 4a, lanes 1 and 3). We did not detect the presence of the Cox 4 subunit of the cytochrome oxidase in this strain (Fig. 4a, lane 3).