A genetic
approach to identify components involved in phosphatidylethanolamine homeostasis
in yeast mitochondria.
Ruth Nebauer,
Ruth Birner, Günther Daum
Institute of Biochemistry, Technical University Graz, Petersgasse 12/II, Graz,
8010, Austria (ruth.nebauer@tugraz.at)
Phosphatidylethanolamine
(PtdEtn) is an essential component of yeast mitochondrial membranes. Psd1p, the
major yeast phosphatidylserine decarboxylase which produces PtdEtn, is a
component of the inner mitochondrial membrane. Since phosphatidylserine
(PtdSer), the precursor of PtdEtn, is formed in the endoplasmic reticulum,
import of PtdSer into mitochondria is an important process. When mitochondrial
Psd1p is deleted, PtdEtn can only be formed in the extramitochondrial space by
Psd2p or through the CDP-ethanolamine pathway. Under these conditions import of
PtdEtn into mitochondria becomes essential for mitochondrial function. To
identify components which are involved in PtdSer transport between the
endoplasmic reticulum and mitochondria we performed screenings under different
conditions. Using the mTn3-lacZ/LEU2 insertion library we mutagenized a psd2 strain thereby
generating insertion/disruption alleles by homologous recombination. We
screened more than 100,000 colonies for cold- and temperature-sensitive
ethanolamine auxotrophy on glucose minimal medium. The genomic sites of
transposon insertion were determined by vectorette PCR, which led to the
identification of several mutations which appear to cause depletion of
mitochondrial PtdEtn. The role of the respective gene products in PtdEtn
biosynthesis and/or interorganelle transport of PtdSer or PtdEtn is subject of
present investigations. (Supported by the FWF project 14468 to G.D.)