Molecular species of phosphatidylinositol-cycle intermediates in the endoplasmic reticulum and plasma membrane - PubMed (original) (raw)

Molecular species of phosphatidylinositol-cycle intermediates in the endoplasmic reticulum and plasma membrane

Yulia V Shulga et al. Biochemistry. 2010.

Abstract

Phosphatidylinositol (PI) turnover is a process requiring both the plasma and ER membranes. We have determined the distribution of phosphatidic acid (PA) and PI and their acyl chain compositions in these two subcellular membranes using mass spectrometry. We assessed the role of PI cycling in determining the molecular species and quantity of these lipids by comparing the compositions of the two membranes isolated from embryonic fibroblasts obtained from diacylglycerol kinase epsilon (DGKepsilon) knockout (KO) and wild-type (WT) mice. In the KO cells, the conversion of arachidonoyl-rich DAG to PA is blocked by the absence of DGKepsilon, resulting in a reduction in the rate of PI cycling. The acyl chain composition is very similar for PI and PA in the endoplasmic reticulum (ER) versus plasma membrane (PM) and for WT versus KO. However, the acyl chain profile for PI is very different from that for PA. This indicates that DGKepsilon is not facilitating the direct transfer of a specific species of PA between the PM and the ER. Approximately 20% of the PA in the ER membrane has one short acyl chain of 14 or fewer carbons. These species of PA are not converted into PI but may play a role in stabilizing regions of high positive curvature in the ER. There are also PI species in both the ER and PM for which there is no detectable PA precursor, indicating that these species of PI are unlikely to arise via the PI cycle. We find that in the PM of KO cells the levels of PI and of PA are decreased approximately 3-fold in comparison with those in either the PM of WT cells or the ER of KO cells. The PI cycle is slowed in the KO cells; hence, the lipid intermediates of the PI cycle can no longer be interconverted and are depleted from the PI cycle by conversion to other species. There is less of an effect of the depletion in the ER where de novo synthesis of PA occurs in comparison with the PM.

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Figures

Fig. 1

Fig. 1

Scheme of PI-cycle.

Fig. 2

Fig. 2

Isolation of PM and ER membrane fractions by iodixanol gradient centrifugation. Fractionation was performed using a 3–25% OptiPrep gradient, and fractions were analyzed by immunoblotting with antibodies against GRP-94 (ER marker) and Na/K ATPase α (PM marker). PNS – postnuclear supernatant.

Fig. 3

Fig. 3

Comparison of ratios of PA or PI in the PM to ER for DGKε KO and WT cells. Results are presented as a mean of PM/ER ratio ± SEM. In the KO case, all PM/ER ratios shown are significantly less than one with p < 0.05 except for PA 32:0 (p=0.06), PA 36:0 (p=0.08), PA 38:3 (p=0.08), and PI 34:0 (p=0.19). The only PM/ER ratios in the WT case which are significantly less than one are PI 36:1 (p=0.01) and PI 36:2 (p=0.03).

Fig. 4

Fig. 4

Comparison of ratios of PA and PI in DGKε KO to WT cells in plasma and ER membranes. Results are presented as a mean of KO/WT ratio ± SEM. Values statistically differed with p < 0.05 are labelled with asterisks.

Fig. 5

Fig. 5

Ratio of PI to PA in the plasma and ER membranes of DGKε WT cells. Results are presented as a mean of PI/PA ratio ± SEM. All ratios are statistically different from 1.0 with p<0.05 except for 32:1 and 34:0 in the PM.

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