GBF1-Arf-COPI-ArfGAP-mediated Golgi-to-ER Transport Involved in Regulation of Lipid Homeostasis

Abstract

ABSTRACT. Eukaryotic cells store neutral lipids and cholesteryl esters in cytoplasmic lipid droplets (LDs), which are generated from the endoplasmic reticulum (ER). Accumulating lines of evidence have indicated that Golgi-to-ER-retrograde transport mediated by COPI-coated vesicles under the control of Arf small GTPases is implicated in LD formation and utilization. However, the detailed mechanism underlying the regulation of lipid homeostasis by COPI-dependent transport has been poorly understood. Here we show that LD deposition and the cellular triacylglycerol content are significantly increased by siRNA-mediated depletion of not only β-COP (a subunit of the COPI coat complex) but also GBF1 (a guanine nucleotide exchange factor for Arfs), Arf4 and Arf5 (class II Arfs), and ArfGAP1-ArfGAP3 (GTPase-activating proteins for Arfs). Although a previous proteomic study suggested the presence of COPI subunits and Arfs on LDs, we have failed to show that components of the GBF1-Arf-COPI-ArfGAP retrograde transport machinery are directly associated with and closely apposed to LDs. Furthermore, although recent studies suggested that COPI-mediated transport and GBF1 participated in delivery of adipose triglyceride lipase (ATGL) onto the LD surface, we have found that depletion of β-COP or GBF1 does not affect association of ATGL with LDs or ATGL-mediated lipolysis. On the basis of these results, we propose other mechanisms how the GBF1-Arf-COPI-ArfGAP transport machinery is implicated in the regulation of lipid homeostasis.

COPI-mediated retrieval of SCAP is critical for regulating lipogenesis under basal and sterol-deficient conditions

“…Previous studies by our group and others have shown that a block in Golgi-to-ER retrograde trafficking through COPI-coated vesicles causes an increase in lipid deposition (Beller et al, 2008;Guo et al, 2008;Soni et al, 2009;Takashima et al, 2011). Here, we extended our previous study and showed that aberrant activation of SREBPs, master regulators of biosynthesis of fatty acids and cholesterol, can be ascribed, at least in part, to the increased lipid deposition that occurs under COPI-compromised conditions.…”

“…Furthermore, COPI-dependent delivery of ATGL to lipid droplets has been implicated in lipolysis, suggesting that inhibition of COPI-mediated trafficking results in lipid storage (Ellong et al, 2011;Soni et al, 2009). We have recently confirmed that knockdown of COPImediated trafficking components results in increased lipid storage; however, we failed to show direct evidence for the involvement of COPI-mediated trafficking in the delivery of ATGL to lipid droplets (Takashima et al, 2011). Therefore, we were interested in lipogenesis as well as lipolysis in order to understand the role of COPI-mediated trafficking in lipid homeostasis.…”

“…We have previously shown that cells depleted of any component of COPI-mediated trafficking, including β-COP (also known as COPB1), exhibit enlarged lipid droplets associated with an increase in cellular TAG content (Takashima et al, 2011); COPIcoated vesicles are involved in retrograde protein trafficking from the cis-Golgi to the ER, whereas COPII-coated vesicles are involved in anterograde trafficking of proteins, including the transport of the SCAP-SREBP complex, from the ER to the Golgi (Harter and Wieland, 1996). This prompted us to examine whether SREBPs, which are master regulators of TAG and cholesterol biosynthesis, are implicated in the observed increase in cellular lipid storage, for the following reasons -(i) ER-to-Golgi trafficking regulates the activation of SREBPs; under sterol-depleted conditions, SCAP escorts SREBPs to the Golgi through COPII-coated vesicles, and at the Golgi, SREBPs undergo sequential processing by the Golgiresident S1P and S2P endoproteases to liberate the cytosolic TFDs (Goldstein et al, 2006;Horton et al, 2002) (also see Fig.…”

“…1A,B), as described previously (Saitoh et al, 2009;Takashima et al, 2011). We then incubated these cells in medium containing lipoproteindeficient serum (LPDS) and oleic acid that had been supplemented with or without 25-hydroxycholesterol (25-HC), which blocks the Fig.…”

“…This hypothesis was based on the fact that the treatment of cells with brefeldin A (BFA), which causes the Golgi complex to collapse into the ER by inhibiting Arf guanine nucleotide exchange factors (Jackson, 2000;Sciaky et al, 1997), results in increased processing of SREBP2 owing to mislocalization of S1P to the ER , as well as in increased lipid deposition and TAG content (Beller et al, 2008;Takashima et al, 2011). As shown in supplementary material Fig.…”