Search

Recent studies showed that the phospholipase subunits of Platelet Activating Factor Acetylhydrolase (PAFAH) Ib, α1 and α2 partially localize to the Golgi complex and regulate its structure and function. Using siRNA knockdown of individual subunits, we find that α1 and α2 perform overlapping and unique roles in regulating Golgi morphology, assembly, and secretory cargo trafficking. Knockdown of either α1 or α2 reduced secretion of soluble proteins, but neither single knockdown reduced secretion to the same degree as knockdown of both. Knockdown of α1 or α2 inhibited reassembly of an intact Golgi complex to the same extent as knockdown of both. Transport of VSV-G was slowed but at different steps in the secretory pathway: reduction of α1 slowed trans Golgi network to plasma membrane transport, whereas α2 loss reduced endoplasmic reticulum to Golgi trafficking. Similarly, knockdown of either subunit alone disrupted the Golgi complex but with markedly different morphologies. Finally, knockdown of α1, or double knockdown of α1 and α2, resulted in a significant redistribution of kinase dead protein kinase D from the Golgi to the plasma membrane, whereas loss of α2 alone had no such effect. These studies reveal an unexpected complexity in the regulation of Golgi structure and function by PAFAH Ib. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Previous studies have shown that membrane tubule-mediated export from endosomal compartments requires a cytoplasmic phospholipase A(2) (PLA(2)) activity. Here we report that the cytoplasmic PLA(2) enzyme complex platelet-activating factor acetylhydrolase (PAFAH) Ib, which consists of α1, α2, and LIS1 subunits, regulates the distribution and function of endosomes. The catalytic subunits α1 and α2 are located on early-sorting endosomes and the central endocytic recycling compartment (ERC) and their overexpression, but not overexpression of their catalytically inactive counterparts, induced endosome membrane tubules. In addition, overexpression α1 and α2 altered normal endocytic trafficking; transferrin was recycled back to the plasma membrane directly from peripheral early-sorting endosomes instead of making an intermediate stop in the ERC. Consistent with these results, small interfering RNA-mediated knockdown of α1 and α2 significantly inhibited the formation of endosome membrane tubules and delayed the recycling of transferrin. In addition, the results agree with previous reports that PAFAH Ib α1 and α2 expression levels affect the distribution of endosomes within the cell through interactions with the dynein regulator LIS1. These studies show that PAFAH Ib regulates endocytic membrane trafficking through novel mechanisms involving both PLA(2) activity and LIS1-dependent dynein function.

We report that platelet-activating factor acetylhydrolase (PAFAH) Ib, comprised of two phospholipase A(2) (PLA(2)) subunits, alpha1 and alpha2, and a third subunit, the dynein regulator lissencephaly 1 (LIS1), mediates the structure and function of the Golgi complex. Both alpha1 and alpha2 partially localize on Golgi membranes, and purified catalytically active, but not inactive alpha1 and alpha2 induce Golgi membrane tubule formation in a reconstitution system. Overexpression of wild-type or mutant alpha1 or alpha2 revealed that both PLA(2) activity and LIS1 are important for maintaining Golgi structure. Knockdown of PAFAH Ib subunits fragments the Golgi complex, inhibits tubule-mediated reassembly of intact Golgi ribbons, and slows secretion of cargo. Our results demonstrate a cooperative interplay between the PLA(2) activity of alpha1 and alpha2 with LIS1 to facilitate the functional organization of the Golgi complex, thereby suggesting a model that links phospholipid remodeling and membrane tubulation to dynein-dependent transport.