J. Biochem, 2001, Vol. 129, No. 1 19-26
© 2001 Japanese Biochemical Society
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Intracellular Transport of Phosphatidic Acid and Phosphatidylcholine into Lipid Bodies in an Oleaginous Fungus, Mortierella ramanniana var. angulispora1
Applied Microbiology Department, National Institute of Bioscience and Human Technology Tsukuba, Ibaraki 305-8566
2To whom correspondence should be addressed. Tel: +81-298-61-6163, Fax: +81-298-61-6172, E-mail: kamisaka{at}nibh.go.jp
Exogenous fluorescent phosphatidic acid (PA) and phosphatidylcholine (PC) were transported into lipid bodies in an oleaginous fungus, Mortierella ramanniana var. angulispora [Kamisaka et al. (1999) Biochim. Biophys. Acta 1438, 185–198]. We further investigated the processes of fluorescent PA and PC transport into lipid bodies in this fungus by changing culture conditions. Lowering incubation temperature decreased lipid body labeling by 1-palmitoyl, 2-[5-(5, 7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-PA (C5-DMB-PA), but fluorescence did not accumulate in organelles other than lipid bodies. C5-DMB-PC transport into lipid bodies was blocked at temperatures below 15°C and fluorescence accumulated in intracellular membranes, presumably endoplasmic reticulum membranes. The low-temperature block of C5-DMB-PC transport enabled us to do pulse-chase experiments in which fungal cells were pulse-labeled at 15°C with C5-DMB-PC and chased at 30°C. The results clearly depicted transport of C5-DMB-PC and its derivatives from intracellular membranes to lipid bodies. Transport was temperature-dependent and ATP-dependent, although microtubules and actin filaments were not substantially involved. Experiments using 14C-labeled fatty acids and glycerol instead of C5-DMB-PC under the same conditions suggested that transport depicted by fluorescence agreed with metabolism and transport of PC containing native fatty acids. Furthermore, the transport mechanism preferred PC containing unsaturated fatty acids such as linoleic acid. This study dissect lipid transport of PA and PC into lipid bodies and reveal regulatory steps for lipid body formation in this fungus.
1This work was supported in part by a Special Coordination Fund for Promoting Science and Technology from the Science and Technology Agency of Japan.
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