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

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.

SUBMITTER: Shulga YV

PROVIDER: S-EPMC2805767 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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

Shulga Yulia V YV Myers David S DS Ivanova Pavlina T PT Milne Stephen B SB Brown H Alex HA Topham Matthew K MK Epand Richard M RM

Biochemistry 20100101 2

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) ...[more]

PMID: 20000336

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Project description:Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated ?-super-tether (?-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in ?-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in ?-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of ?-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Dataset Information

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

Publications

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

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