Misregulation of a DDHD Domain-containing Lipase Causes Mitochondrial Dysfunction in Yeast

Abstract

The DDHD domain-containing proteins, which belong to the intracellular phospholipase A 1 (iPLA 1 ) family, have been predicted to be involved in phospholipid metabolism, lipid trafficking, membrane turnover, and signaling. Defective cardiolipin (CL), phosphatidylethanolamine, and phosphatidylglycerol remodeling cause Barth syndrome and mitochondrial dysfunction. Here, we report that Yor022c is a Ddl1 (DDHD domaincontaining lipase 1) that hydrolyzes CL, phosphatidylethanolamine, and phosphatidylglycerol. Ddl1 has been implicated in the remodeling of mitochondrial phospholipids and CL degradation. Our data also suggested that the accumulation of monolysocardiolipin is deleterious to the cells. We show that Aft1 and Aft2 transcription factors antagonistically regulate the DDL1 gene. This study reveals that the misregulation of DDL1 by Aft1/2 transcription factors alters CL metabolism and causes mitochondrial dysfunction in the cells. In humans, mutations in the DDHD1 and DDHD2 genes cause specific types of hereditary spastic paraplegia (SPG28 and SPG54, respectively), and the yeast DDL1-defective strain produces similar phenotypes of hereditary spastic paraplegia (mitochondrial dysfunction and defects in lipid metabolism). Therefore, the DDL1-defective strain could be a good model system for understanding hereditary spastic paraplegia.

Cardiolipin deficiency causes triacylglycerol accumulation in Saccharomyces cerevisiae

“…9; Building N250, D-60438 Frankfurt, Germany). The pYES2/NT B-DDL1 (pRP1) and pYES2/NT C-DGA1 (pRK13) plasmids were described previously [18,19]. The yeast strains used in this work were stored as glycerol stocks at -80°C.…”

“…An equal quantity of cells (A 600 = 25) was collected for lipid extraction. Bligh and Dyer's method was used with slight modifications for the lipid extraction as reported in a previous study [18]. CL was separated from other phospholipids on a TLC plate using a two-dimensional solvent system (1st dimension, chloroform:methanol:25% ammonia, 65:35:4, v/v and 2nd dimension, chloroform:methanol:acetic acid:water, 85:25:5:4, v/v), followed by phosphorimaging.…”

“…The His-tagged Dga1 protein was expressed according to the protocol used in the previous studies [18,22]. Briefly, cells harboring the pYES2/NT C-DGA1 construct were induced in the rich induction medium (3 9 YPG, 3% yeast extract, 6% peptone, and 6% galactose, pH 6.5) to produce a higher yield of the protein.…”

“…The analysis of the molecular species of TAG was performed using the MS/MS ALL technique [18,24,25]. Briefly, the stationary phase yeast cells were harvested and lyophilized, and 10 mg of the lyophilized cells was taken for lipid extraction.…”

“…Total RNA isolation was performed using an RNA isolation kit (NucleoSpinÒ RNA II kit, Macherey-Nagel), and a high-capacity cDNA reverse transcription kit (Applied Biosystems) was used for the complementary DNA (cDNAs) synthesis. The real-time quantitative PCR (qRT-PCR) analyses were performed according to the procedure reported previously [18]. The primers used in the transcripts analyses are listed in Table 2.…”