Phospholipid Synthesis Participates in the Regulation of Diacylglycerol Required for Membrane Trafficking at the Golgi Complex

Abstract

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the postGolgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.

Structural Basis for Autoinhibition of CTP:Phosphocholine Cytidylyltransferase (CCT), the Regulatory Enzyme in Phosphatidylcholine Synthesis, by Its Membrane-binding Amphipathic Helix

“…This version was used to incorporate a fluorophore at one site in that work and has near-WT activity (21). This construct also has an N-terminal His tag that adds the sequence MAKH 6 IEGRSA before the first methionine of CCT. Using pAX142 HisCCT-367(S288C) as template, we created pAX142 versions of CCT-367(6S) and CCT-367(6A) by making serine or alanine substitutions at codons 285, 286, 289, 290, 292, and 293 using mutagenic primers and QuikChange mutagenesis.…”

“…It catalyzes the rate-limiting step in PC synthesis, the formation of the headgroup donor, CDP-choline. In addition to its role in maintaining membrane phospholipid compositional balance, targeted gene deletions or siRNA-mediated suppression have revealed the involvement of the major isoform, CCT␣, in lung surfactant production (3), plasma lipoprotein balance (4), size regulation of lipid storage droplets (5), and control of diacylglycerol concentration in the Golgi, which in turn regulates vesicular traffic (6).…”

Lipid phosphate phosphatase 3 participates in transport carrier formation and protein trafficking in the early secretory pathway

“…Moreover, there is a constant and relatively large pool of DAG in Golgi membranes (Lev, 2006). For all these reasons, DAG has been extensively associated with vesicular transport events at the Golgi in yeast and mammalian cells (Asp et al, 2009;Baron and Malhotra, 2002;Fernández-Ulibarri et al, 2007;Kearns et al, 1997;Litvak et al, 2005;Sarri et al, 2011). Nonetheless, PA has recently been reported to regulate transport carrier formation at the Golgi (Yang et al, 2008;Yang et al, 2011).…”

A high throughput siRNA screen identifies genes that regulate mannose 6-phosphate receptor trafficking

“…Knockdown of the 5-phosphatase INPP5J, of the kinase PIK3CB or of PIK3AP1 caused the accumulation of GFP-MPR at the cell surface, indicating a perturbation of PIP metabolism at the plasma membrane and an impaired GFP-MPR endocytosis. PIP metabolism has been shown to be coupled to the production of diacylglycerol (DAG) (Sarri et al, 2011) and phosphatidic acid, two cone-shaped lipids inducing membrane curvature. Depletion of the phosphatidic acid phosphatase type 2A (PPAP2A) caused the dispersion of the perinuclear GFP-MPR compartment ( Fig.…”