Ypr140wp, 'the yeast tafazzin', displays a mitochondrial lysophosphatidylcholine (lyso-PC) acyltransferase activity related to triacylglycerol and mitochondrial lipid synthesis

Abstract

When the yeast protein Ypr140w was expressed in Escherichia coli, a lyso-PC [lysophosphatidylcholine (1-acylglycerophosphorylcholine)] acyltransferase activity was found associated with the membranes of the bacteria. To our knowledge, this is the first identification of a protein capable of catalysing the acylation of lyso-PC molecules to form PC. Fluorescence microscopy analysis of living yeasts revealed that the fusion protein Ypr140w-green fluorescent protein is targeted to the mitochondria. Moreover, in contrast with wild-type cells, in the absence of acyl-CoA, the yeast mutant deleted for the YPR140w gene has no lyso-PC acyltransferase activity associated with the mitochondrial fraction. When yeast cells were grown in the presence of lactate, the mutant synthesized 2-fold more triacylglycerols when compared with the wild-type. Moreover, its mitochondrial membranes contained a lesser amount of PC and cardiolipin, and the fatty acid composition of these latter was greatly changed. These modifications were accompanied by a 2-fold increase in the respiration rates (states 3 and 4) of the mitochondria. The relationship between the deletion of the YPR140w gene and the lipid composition of the ypr140wDelta cells is discussed.

Figure 1. Amino acid sequence alignment of S. cerevisiae Ypr140wp with various tafazzin proteins

Proteins other than Ypr140wp are tafazzins from Neurospora crassa (Neucr;TAZ; EAA29034), human (Homsa;TAZ; AAO84344), drosophila (Drome;TAZ; AAD48409), mouse (Musmu;TAZ; AAH15305) and Caenorhabditis elegans (Caeel;TAZ; CAA92638). Identical amino acids are represented by dots. Sequences were compared using the multiple alignment program CLUSTAL W version 1.7. The tafazzin signature is boxed and the phospholipid and glycerol acyltransferase motifs are in boldface letters.

Figure 2. Mitochondrial location of Ypr140wp

ypr140wΔ cells expressing the Ypr140w–GFP fusion protein were grown in YNB medium supplemented with 2% lactate. A total of 10⁷ cells were then stained with 5 μM DASPMI and examined under a fluorescence microscope as described in the Experimental section.

Figure 3. Lyso-PC acyltransferase activity associated with membranes of E. coli expressing Ypr140wp and induction by IPTG of Ypr140w–HA fusion protein synthesis

Upper panel, immunoblot experiments were performed by using rabbit polyclonal antibody to HA as described in the Experimental section (10 μg of lysates per lane). Experimental details are as given in the legend to Figure 2, except that pET-15b contained the coding sequence of the Ypr140w–HA fusion protein instead of Ypr140wp. Lower panel, lyso-PC acyltransferase activities were determined using 1 nmol of labelled lyso-PC and 100 μg of membrane proteins as described in the Experimental section. Membrane proteins were obtained from C41 (DE3) E. coli (A), C41 (DE3) E. coli transformed with pET-15b (B), E. coli transformed with pET-15b containing the coding sequence of Ypr140wp [clone 1 (C) and clone 2 (D)]. The protein synthesis was induced (+) or not (−) by IPTG.

Figure 4. Acyl-CoA-independent lyso-PC-acyltransferase activity associated with mitochondria purified from ypr140wΔ mutant and wild-type cells

Lyso-PC acyltransferase activities were determined for 30 min using 0.5 nmol of labelled lyso-PC and various amounts of mitochondria purified from mutant (closed symbols) or wild-type (open symbols) cells. Results are representative of three independent experiments.

Figure 5. Phospholipid composition of ypr140wΔ mutant and wild-type cells

Mutant (closed bars) or wild-type (open bars) cells were grown in a medium supplemented with either 2% glucose (A, B) or 2% lactate (C, D), and harvested during the exponential (A, C) or stationary (B, D) phase. Lipids were purified and quantified as described in the Experimental section. The error bars represent the means±S.D. for three determinations. PS, phosphatidylserine; Cardio, cardiolipin.

Scheme 1. Possible explanation for the lipid phenotype of ypr140wΔ cells

For more clarity, the TAG synthesis pathway occurring in lipid bodies is not shown. + (−) represents the rates that might increase (decrease) or tend to increase (decrease) in ypr140wΔ cells. CL, cardiolipin.