Lipids and Golgi function: An acyltransferase stops traffic

Lipidomics Gateway (26 August 2009) [doi:10.1038/lipidmaps.2009.21]

A lysophosphatidic acid-specific acyltransferase enzyme regulates Golgi morphology and function.

The enzymatic production and interconversion of lipid species that favor membrane curvature are implicated in membrane events including the formation of vesicles and tubules. In the Golgi, phospholipase A₂-mediated generation of lysophospholipids (LPLs) is involved in retrograde trafficking and membrane tubule formation. However, Golgi phospholipid remodeling has not been entirely characterized. Now in the Journal of Cell Biology, Schmidt and Brown report the identification of the integral membrane enzyme 1-acylglycerol-3-phosphate O-acyltransferase 3 (AGPAT3, also known as lysophosphatidic acid acyltransferase-3, LPAAT3, or LPAAT-γ) which opposes the phospholipase A₂-mediated effects in the regulation of Golgi structure, trafficking and tubulation.

Membrane lipids that favor negative curvature — because their acyl chain region is more bulky than the headgroup — include phosphatidic acid (PA) and diacylglycerol (DAG). Conversely, lysophospholipids have only one acyl chain and induce positive membrane curvature. Phospholipids are generated from LPLs by the transfer of a fatty acid chain from an acyl-CoA donor onto the LPL acceptor, by an acyltransferase enzyme. Two ER-resident acyltransferases that have lysophosphatidic acid (LPA) as a preferred acceptor have been characterized, but nine such enzymes are annotated in the human genome. To find out if any might be involved in Golgi function, Schmidt and Brown expressed GFP-tagged versions of four uncharacterized candidates in cultured cells. One, AGPAT3, colocalized with Golgi markers.

An assay for the production of radiolabelled phospholipid products using ¹⁴C-palmitoyl CoA and various LPL species showed that AGPAT3 does indeed have LPA acyltransferase activity, and that this activity increases the level of PA in the Golgi. To investigate the effect of AGPAT3 expression on Golgi structure and function, the authors used a combination of stable overexpression of the protein, short interfering RNA (siRNA)-mediated knockdown, and expression of an siRNA-resistant construct. Reduced AGPAT3 expression resulted in fragmentation of the Golgi and the formation of mini-stacks. Overexpression of the protein reduced the rate, number and length of Golgi tubules formed in response to stimulation, both in cultured cells and in vitro.

Influencing Golgi tubulation suggests a role in Golgi trafficking. When cells are incubated at 15°C, an endogenous marker involved in retrograde trafficking accumulates in the cis-Golgi, and returns rapidly to the ER via membrane tubules when the temperature is shifted to 37°C. In cells overexpressing AGPAT3, this retrograde recycling was delayed, whereas in knockdown cells it proceeded more quickly. Likewise, anterograde membrane trafficking was affected by expression of AGPAT3: the extracellular activity of a soluble, secreted cargo co-transfected with the other constructs was increased in the AGPAT3 knockdown cells and reduced in the overexpressing cells.

The results of this study confirm the importance of phospholipid remodeling in the maintenance of Golgi structure, and in the formation of membrane trafficking intermediates. Characterization of other phospholipase and acyltransferase enzymes will help to define the role of curve-inducing lipids in all membrane trafficking pathways.

Emma Leah