PAFAH Ib phospholipase A2 subunits have distinct roles in maintaining Golgi structure and function

Abstract

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.

Figure 1

Single knockdown of either PAFAH Ib α1 or α2 causes differential fragmentation of the Golgi complex. (A) Western blots showing amounts of α1 and α2 in control, single, or double siRNA-treated BTRD cells 72 h after initial siRNA transfection. Dynamin served as a loading control. (B) Confocal z-series of the Golgi (anti-ManII immunofluorescence). (C) The morphological changes in Golgi structure were quantified ‘blind’, counted as normal Golgi (intact), webbed Golgi ribbons (α1 siRNA panel on the left) or fragmented (α1 siRNA panel on the right). (D) Quantification of fragmented Golgi, subcategorized into fragmented (not swollen or shrunken), dilated (swollen), and vesiculated (shrunken). (E) Direct wide-field fluorescence of ERGIC53-GFP in control, single, and double knockdowns. Scale bars = 5 μm. (F) Transmission electron microscopy of thin sections from control and single knockdown BTRD cells. Scale bars = 500 nm.

Figure 2

Secretory protein trafficking is inhibited at different steps in single PAFAH Ib α1 or α2 knockdown cells. (A) Kinetics of ssHRP secretion, measured by HRP activity in the media of control or siRNA-treated (as indicated) BTRD cells. Values were normalized to expression levels of HRP and HRP activity in control cells at 8 h. (B) Confocal images of ts045 VSV-G-YFP transport in single knockdown cells. Transfected cells were incubated at 40°C to accumulate ts045 VSV-G-YFP in the ER, shifted to 20°C for 20 or 120 min to allow export from the ER and transport to the TGN, and then shifted to 32°C for 80 min to allow export from the TGN and transport to the cell surface. (C) Quantification of the percent of total ts045 VSV-G-YFP fluorescence on the PM following shift to 32°C for 80 min. For α2 knockdown cells, only cells with VSV-G outside the ER (~80% of cells) were measured.

Figure 3

Localization of vesicle markers and protein kinase D in single knockdown cells. (A) Knockdown of PAFAH Ib α1 or α2 does not detectably affect clathrin AP-1 adaptor, COPI subunit β-COP, or the COPII subunit Sec31a in BTRD cells. Scale bars = 5 μm for AP1 and β-COP, 10 μm for Sec31a. (B) Knockdown of α1, but not α2, causes a loss of PKD-KD-GFP from the TGN and redistribution to the plasma membrane. Scale bars = 10 μm.

Figure 4

Golgi reassembly following BFA washout is inhibited in PAFAH Ib α1 or α2 single knockdown cells. (A) Confocal images of control and cells treated with siRNA against individual or both subunits. BTRD cells were treated with media containing 5 μg/mL BFA for 20 min, followed by replacement with BFA-free media to allow reassembly of the Golgi. Cells were fixed at the indicated time points (after BFA removal) and processed for immunofluorescence with anti-ManII. Scale bars = 10 μm. (B) Quantification of reassembly into an intact Golgi ribbon in control and siRNA-treated cells. Error bars = SEM. (C) Confocal micrographs illustrating the morphology of the Golgi complex (anti-ManII immunofluorescence) during recovery from BFA in control and siRNA treated cells. Scale bars = 2 μm.