The manipulation of cellular cytochrome and lipid composition in a haem mutant of Saccharomyces cerevisiae

Abstract

1. The ole-3 mutant of Saccharomyces cerevisiae has an early lesion in the pathway of porphyrin biosynthesis. 2. This results in the loss ofall haem-containing enzymes, including the mitochondrial cytochromes, and prevents the synthesis of components whose formation requires haem-containing enzymes, including unsaturated fatty acids, ergosterol and methionine. 3. The pleiotropic effects of the primary lesion are reversed by growing mutant ole-3 aerobically in the presence of intermediates of the porphyrin-biosynthetic pathway, and the present work reports the degree ofmanipulation oflipid and respiratorycytochrome composition. 4. Supplements of 5-aminolaevulinate in the range 0.5-500mg/l result in a progressive increase in the cellular content of unsaturated fatty acids and respiratory cytochromes, cause the replacement of lanosterol and squalene by ergosterol, and an increase in total sterol content. 5. Haematoporphyrin and protoporphyrin IX have similar but less extensive effects on cellular composition, whereas haematin allows unsaturated fatty acid synthesis and some sterol synthesis, but has no effect on the formation of respiratory cytochromes. 6. These results suggest that growth of the organism in the presence of defined amounts of 6-aminolaevulinate will be useful in the investigation of the role of lipids and cytochromes in the function and assembly of mitochondrial membranes.

Biochemical Basis of Oxidative Protein Folding in the Endoplasmic Reticulum

“…To identify novel binding partners of p-arrestin 2, we used the yeast two-hybrid system based on a GAL4-6-arrestin 2 fusion protein to screen a rat brain cDNA libran (13). Three positive clones were obtained, two of which encoded fusion proteins of the GAL4 activation domain with a COOH-terminal portion of JNK2 and one that encoded a COOH-terminal portion of the JNK3 isoform (14).…”

“…Deoxidizes the CxxC active site of the PDI letion of COQ5 or HEMI, which blocks biohomolog DsbA, which then catalyzes disulsynthesis of ubiquinone (11,12) or heme fide formation in folding proteins. DsbB is (13), respectively, inhibited respiration and reoxidized by ubiquinone produced during ER-associated cytochromes but did not alter respiration.…”

Regulation of ergosterol biosynthesis and sterol uptake in a sterol-auxotrophic yeast

“…Hemoproteins are known to participate in several steps of the sterol biosynthetic pathway, including dehydrogenation and demethylation reactions (13,21). Thus, sterol biosynthesis is precluded in anaerobically cultured wild-type cells or in heme-deficient mutants cultured aerobically because of lack of the corresponding hemoproteins (2,3,12). Previous studies have indicated that when wild-type cells are grown aerobically, they are apparently impermeable to exogenous sterols (17,27).…”

The Effects of Catabolite Derepression on the Accumulation of Steryl Esters and the Activity of -Hydroxymethylglutaryl-CoA Reductase in Saccharomyces cerevisiae

“…Bailey & Parks (1975) showed that zymosteryl esters are not methylated by the Cea methyltransferase in vitro, and its seems likely that the other steryl esters are unavailable for direct biosynthetic modification. Under anaerobic conditions, the steryl esters appear to be converted to the free sterol, of which zymosterol and fecosterol are then completely converted to ergosterol and 24 : 28-dehydroergosterol, but lanosterol remains as such because of the absence of molecular oxygen.…”

“…A calibration curve was prepared using bovine serum albumin (Calbiochem, Type V). Dry weights of cells were determined volumetrically by centrifuging a sample of cells in calibrated thick-walled graduated glass capillary tubes (Astin et al, 1977). Ethanol was assayed using the g.1.c.…”