Graz University of Technology Austria Institute of Biochemistry ...

However, mechanisms involved in these translocation processes are still unclear. To address
these questions, we established in vitro and in vivo assays for studying phospholipid supply
to peroxisomal membranes. We constructed a strain which lacks the gene product of OPI3,
the major PE methyltransferase localized to the ER, and bears an Opi3p-GFP hybrid with an
SKL targeting sequence that directs the enzyme to peroxisomes. In this “reporter mutant”,
the only site of phosphatidylcholine (PC) formation via methylation of PE are the
peroxisomes, and the appearance of PC becomes an indicator and measure for PE
translocation from the different sites of synthesis to peroxisomes. Currently permeabilized
cells of the “reporter mutant” are used to characterize the PE transport to peroxisomes in
some detail. This system in combination with mutations in the different PE biosynthetic
pathways will allow us to investigate the different mechanisms of PE translocation between
organelles involved in aminoglycerophospholipid biosynthesis.
Most recently, we also studied the link between PE metabolism and neutral lipid storage. For
this purpose we analyzed lipids from strains bearing defects in PE synthesis. These analyses
showed that a mutant bearing a defect in the CDP-ethanolamine pathway had a decreased
level of triacylglycerols (TAG). In cki1∆dpl1∆eki1∆ mutants bearing defects in the CDPethanolamine
pathway both the cellular and the microsomal levels of PE were markedly
decreased, whereas in other mutants of PE biosynthetic routes depletion of this
aminoglycerophospholipid in microsomes was less pronounced. This observation is
important because the TAG synthesisizing enzyme Lro1p similar to the enzymes of the CDPethanolamine
pathway is a component of the ER. We conclude from these results that in
cki1∆dpl1∆eki1∆ insufficient local supply of PE to Lro1p was a major reason for the strongly
reduced TAG level.
Neutral lipid storage in lipid particles and mobilization
Yeast cells have the capacity to store neutral lipids TAG and STE (steryl esters) in subcellular
structures named lipid particles/droplets. Upon requirement, TAG and STE can be mobilized
and serve as building blocks for membrane biosynthesis. In a long-standing project of our
laboratory, we investigate the characterization of enzymatic steps which lead to the
mobilization of TAG and STE depots.
A major focus of our neutral lipid project was the biochemical characterization of the three
yeast TAG lipases, Tgl3p, Tgl4p and Tgl5p. Previous work from our laboratory had
demonstrated that deletion of TGL3 encoding the major yeast TAG lipase resulted in
decreased mobilization of TAG, a sporulation defect and a changed pattern of fatty acids,
especially increased amounts of C22:0 and C26:0 very long chain fatty acids in the TAG
fraction. To study a possible link between TAG lipolysis and membrane lipid biosynthesis, we
carried out biochemical experiments with wild type and deletion strains bearing defects in
Tgl3p, Tgl4p and Tgl5p. We demonstrated that tgl mutants had a lower level of sphingolipids
and glycerophospholipids than wild type. ESI-MS/MS analyses confirmed that TAG
accumulation in these mutant cells resulted in reduced amounts of phospholipids and
sphingolipids. In vitro and in vivo experiments revealed that TAG lipolysis markedly affected
the metabolic flux of long chain fatty acids and very long chain fatty acids required for
sphingolipid and glycerophospholipid synthesis. The pattern of phosphatidylcholine (PC), PE
and PS molecular species was altered in tgl deletion strains underlining the important role of
TAG turnover in maintenance of the pool size and remodelling of complex membrane lipids.
This study shed new light on the physiological role of TAG lipases in yeast and in general.
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