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.

Retrograde trafficking from the Golgi complex to endoplasmic reticulum (ER) through COPI-coated vesicles has been implicated in lipid homeostasis. Here, we find that a block in COPI-dependent retrograde trafficking promotes processing and nuclear translocation of sterol regulatory element binding proteins (SREBPs), and upregulates the expression of downstream genes that are involved in lipid biosynthesis. This elevation in SREBP processing and activation is not caused by mislocalization of S1P or S2P (also known as MBTPS1 and MBTPS2, respectively), two Golgi-resident endoproteases that are involved in SREBP processing, but instead by increased Golgi residence of SREBPs, leading to their increased susceptibility to processing by the endoproteases. Analyses using a processing-defective SREBP mutant suggest that a fraction of SREBP molecules undergo basal cycling between the ER and Golgi in complex with SREBP cleavage-activating protein (SCAP). Furthermore, we show that SCAP alone is retrieved from the Golgi and moves to the ER after processing of SREBP under steroldeficient conditions. Thus, our observations indicate that COPImediated retrograde trafficking is crucial for preventing unnecessary SREBP activation, by retrieving the small amounts of SCAP-SREBP complex that escape from the sterol-regulated ER retention machinery, as well as for the reuse of SCAP.

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