Yeast cytoplasm

The biggest concern over the use of recombinant microbes is that microbial cell walls constitute a permeability barrier for test compounds. Enzyme inhibitors that cannot accumulate in bacterial or yeast cytoplasm will appear as false nega-... 

The energy dependence of import into mitochondria has been exploited to accumulate large amounts of precursors in an uncoupler-poisoned living cell [93]. Precursor to the 8-subunit of the proton ATPase has been purified from such cells by affinity chromatography on an antibody column, followed by chromatofocussing and isoelectric focussing. After renaturation, this precursor can be correctly processed by the matrix protease and can be imported into mitochondria, but only in the presence of a proteinaceous factor from the yeast cytoplasm [94]. A similar finding has been reported for import of precursors into rat liver mitochondria in which a factor is provided by the reticulocyte lysate [95,96]. 

Bernardi G., Carnevali F., Nicolaieff A., Piperno G., Tecce G. (1968). Separation and characterization of a satellite DNA from a yeast cytoplasmic petite mutant. J. Mol. Biol. 37 493-505. 

Figure 9 Yeast5. cerevisiae cell with small daughter cell bud and proteins of arsenate reduction and transport. Acr2p the yeast cytoplasmic arsenate reductase. Acr3p the potential-driven membrane arsenite efflux protein, equivalent to bacterial ArsB. Ycflp the novel As(III)-3 GSH adduct carrier than transports the adduct complex into the cell vacuole compartment, functioning as an ATPase.

Figure 3.8 summarises some of the observable effects occurring in C. albicans after treatment with a lethal level of candicidin. Leakage of small molecular weight material (represented by potassium ions) follows closely on antibiotic uptake. Precipitation of the yeast cytoplasm (as represented by a rise in cell... 

In the reductive pathway, the Krebs cycle enzymes are assumed to operate as far as a-oxoglutarate, thus forming a linear pathway. A second linear pathway, from oxaloacetate to malate to fumarate to succinate, is suggested to account for the formation of succinic acid [46]. In support of this new pathway are the observations that (/) yeast contains cytoplasmic malate dehydrogenases capable of converting oxaloacetate to malate, (//) several fumarate reductases (FAD-dependent) have been found in the yeast cytoplasm which have high affinity for fumarate and are unable to oxidize succinate [52] and (Hi) succinate is a significant product of fermentation, i.e. an end product . 

Figure 9.8 A schematic representation of the subcellular compartmentalisation of a wine yeast cell. The cell envelope, comprising a ceU waU, periplasm and plasma membrane, surrounds and encases the yeast cytoplasm. The structural organisation of the intraceUnlar miheu, containing organelles such as the nucleus, endoplasmic reticulum, Golgi apparatus, mitochondria and vacuoles, is maintained by a cytoskeleton. Several of these organelles derive from an extended intramembranous system and are not completely independent of each other. Adapted from Pretorius (2000).

When observed under the electron microscope, the yeast cytoplasm appears rich in ribosomes. These tiny granulations, made up of ribonucleic acids and proteins, are the center of protein synthesis. Joined to polysomes, several ribosomes migrate the length of the messenger RNA. They translate it simultaneously so that each one produces a complete polypeptide chain. 

In cell-free systems the inhibition of the transfer of aminoacyl-transfer RNAs to polypeptide (at the ribosome level) is probably the primary effect The most interesting effect of cycloheximide is that protein synthesis by isolated mitochondria of eukaryotic cells, like bacterial ribosomes, but unlike mammalian and yeast cytoplasmic ribosomes, is not inhibited over a wide range of concentrations. Despite this selective action, cycloheximide is extremely harmful to the biogenesis of mitochondria in vivo, due to a large contribution of the microsomal protein synthesizing system in the formation of mitochondrial proteins. 

In order to examine this possibility, the rRNAs from bound 80 S and free cytoplasmic ribosomes were purified and examined by SDS polyacrylamide gel electrophoresis. Under the conditions of electrophoresis (conditions sufficient to completely resolve HeLa, E. coli, yeast cytoplasmic, and intrinsic mitochondrial rRNA), bound and free rRNA coelec-trophorese. This result demonstrates that there is no gross size difference between bound and free rRNA. Both ribosomes contain equal quantities of 5.8 S rRNA, which also coelectrophorese. 

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