The subcellular distribution of carotenoids in Phycomyces blakesleeanus

Abstract

In order to study the subcellular distribution of carotenoids in Phycomyces blakesleeanus, homogenates of mycelia of the C115 carS42 mad-107(-) mutant were fractionated by both differential and density gradient centrifugation. Characterisation of the fractions was achieved by the distribution of marker enzyme activities. Organelles prepared by differential centrifugation were shown to be associated with contaminating lipids, including B-carotene (B, (3-carotene). These lipids could not be removed by either repeated washing, or by centrifugation procedures; so a separation technique based on density gradient centrifugation was devised. Only two fractions isolated by the latter method contained carotenoid; the lipid particles, and a particulate fraction. The latter was tentatively identified, on the basis of its density (1.10g/ml), and its enzyme content, as a vacuole-containing fraction. Both these pigmented fractions exhibited alkaline p-nitro-phenylphosphatase activity, as well as containing sterols and phospholipids. The proteins from these organelles revealed close similarities on amino acid analysis. The relative distribution of B-carotene between these fractions was investigated at various stages of growth. The lipid droplets isolated from P. blakesleeanus resembled those prepared from other plants in both size and lipid content. They contained sufficient protein to form a monolayer around their surf ace. Mycelia were grown in media containing [2-14c]mevalonic acid (MVA), and the subcellular distribution of radioactive lipids was determined. The rates of incorporation of MVA in various organelles were compared. Organelles were also incubated with [2- 14c]MVA in vitro. Cytosol enzymes were able to incorporate MVA into terpenols and squalene. No particulate fractions were capable of converting MVA into (B-carotene or sterols in the absence of the cytosol. These results are discussed in terms of the possible sites of biosynthesis of carotenoids in the mycelium, and the inter-relationships of the pigmented organelles.