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Carotenoid compartments and associated proteins in Blakeslea, Xhanthophyllomyces and Phycomyces

Membrane and globule formation was monitored by light and electron microscopy in Blakeslea. Light microscopy studies using Nomarski optics showed distended hyphal filaments within which coloured compounds accumulated. An EM study of globule formation in the mated strains of F986 and F921 in submerged culture revealed that at 48 h post inoculation, the lipid globules contained internal membranes similar to the prolamellar bodies of etioplasts. At the point of first appearance of colour, 3 days post inoculation, the lipid globules were filled with an extensive internal membrane system not dissimilar to the grana stacks of the thylakoid membrane. These membranous structures then disappeared as the globules increased in size later in development: Globules purified at 7 days post inoculation were devoid of internal membranes and had a greater size distribution.

In Xanthophyllomyces, a large vacuole-like cavity was observed by electron microscopy. The vacuole-like cavity in the mutant strain was observed to occupy a larger proportion of the cell area compared with the wild type strain. No internal membrane systems were found. Protein and transcript levels were not determined.
Globule associated proteins in Blakeslea were separated by SDS-PAGE. At least two proteins, and in some cases three, were found in lipid globules from the wild type strains F921 and F986, and from the mutants SB 38 and SB52. The proteins were less than 30 kDa and highly hydrophobic.

The absence of a known genome hindered protein identification. Poor growth in submerged culture hindered large-scale globule isolation for the purification of sufficient quantities of protein for identification by tandem mass spectrometry and Edmon sequencing.


Peter M. BRAMLEY, (Director of Research)
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